Alopecia areata (AA) is a complex autoimmune disease in which autoreactive T cell-mediated attack of the hair follicle (HF) leads to non-scarring hair loss. Although AA is one of the most prevalent autoimmune diseases, the development of novel effective therapeutics has been limited. Standard of care remains observation for mild cases and steroids for moderate-to-severe cases, which have demonstrated only limited efficacy. The skin is a highly heterogeneous tissue at baseline, comprised of a diverse array of immune and non-immune cell types whose coordinated crosstalk is essential for homeostasis. The skin microenvironment becomes markedly altered as a result of disease-associated inflammation in AA. A pathognomonic histopathologic feature of AA is an intense lymphocytic infiltrate surrounding the lower portion of the HF in the growth phase of the hair cycle, known as anagen. We previously established that CD8+ T cells comprise the majority of this infiltrate in AA skin, and that they are necessary and sufficient to drive disease via JAK/STAT activation. While this discovery led to the pioneering use of JAK inhibitors as a novel class of therapeutics in AA, JAK inhibition is not a curative solution, since patients often experience relapse upon discontinuation of treatment. This not only underscores the continued need for translational drug discovery research in AA, but also reflects an incomplete understanding of the mechanisms that govern disease pathophysiology. Recent advances in single-cell RNA sequencing (scRNAseq) present an unprecedented opportunity to dissect the heterogeneity of complex tissues and disorders. Since its emergence, scRNAseq has proven to be a powerful tool for the discovery of rare cell types and novel therapeutic targets in a variety of contexts that range from cancer to autoimmunity. In this thesis, we leveraged scRNAseq to interrogate the cellular and molecular mechanisms underlying disease pathogenesis in AA at single-cell resolution, together with validation and functional experiments, with the goal of uncovering novel cell types and pathways that can guide the development of innovative therapeutic strategies. In Chapter 2, we performed scRNAseq of skin-infiltrating CD45+ immune cells to dissect lymphocyte heterogeneity in both murine and human AA. Our scRNAseq analyses informed a series of antibody-mediated cell depletion experiments that assessed the in vivo function of specific lymphocyte subsets in murine AA. Our results established CD8+ T cells as the predominant disease-driving cell type in AA. We identified shared mechanisms underlying CD8+ T cell heterogeneity in murine and human AA skin, in which CD8+ T cells form an “effectorness gradient” comprised of interrelated transcriptional states that culminate in increased expression of inflammatory cytokines and T cell effector function. We also demonstrated a role for CD4+ T helper cells in disease initiation, and determined that regulatory T cells possess intact immunosuppressive capacity in AA. In Chapter 3, we expanded upon the studies described in Chapter 2 and performed scRNAseq of skin-infiltrating CD45+ cells at various timepoints throughout disease course (from 3 to 24 weeks post-disease induction) in AA to analyze the temporal dynamics of lymphocyte heterogeneity in AA skin and skin-draining lymph nodes. In conjunction with scRNAseq, we also performed single-cell TCR sequencing to assess the dynamics of T cell clonality alongside changes in T cell transcriptional profiles. We observed a striking increase in CD8+ T cell clonal expansion during disease onset, which increased throughout disease progression and subsequently decreased in chronic AA, when the preclinical mouse model exhibits total body hair loss. Our single-cell analyses suggested that CD8+ T cell clonality and pathogenicity are closely linked, which we validated in vivo by demonstrating that a single expanded clonotypic population of CD8+ T cells is sufficient to induce disease in mice. In Chapter 4, we analyzed single-cell transcriptomic profiles obtained from full-thickness skin in mice with chronic AA to investigate the contributions of the HF and other non-T cell populations in disease. In this study, we also used a network biology-based approach to infer single-cell protein activity, which together with single-cell mRNA gene expression profiles uncovered a multitude of novel findings in AA. Our results revealed a role for necroptosis as a potential HF-intrinsic mechanism of pro-inflammatory signaling in AA, and also identified an MHC Class II signature specific to basal keratinocytes in AA skin. Furthermore, we uncovered a novel, rare population of disease-associated Arg1+ macrophages, which prompted us to revisit our immune-specific scRNAseq datasets described in Chapters 2 and 3 and perform an integrative analysis of this novel cell type in AA. Our preliminary in vivo studies suggested that targeting Arg1+ macrophages and/or arginine metabolism may ameliorate disease in AA. Taken together, this thesis presents a comprehensive, systematic interrogation of AA pathogenesis at single-cell resolution. Importantly, the validation and functional studies that were informed by our scRNAseq data demonstrate proof-of-concept of the use of single-cell technology to accelerate the discovery and translation of novel therapeutic targets in complex diseases. While we undertook a hypothesis-driven approach to design our studies, the data presented in this thesis was also profoundly hypothesis-generating, and has informed a number of ongoing projects in the laboratory with the shared goal of advancing our understanding of disease pathology in AA.