Non-hematopoietic stromal cells (SCs) promote and regulate adaptive immunity through numerous direct and indirect mechanisms. SCs construct and support the secondary lymphoid organs (SLOs) in which lymphocytes crawl on stromal networks and inspect antigen-presenting cells for surface-display of cognate antigens. SCs also secrete survival factors and chemotactic cues that recruit, organize, and facilitate interactions among these leukocytes. They influence antigen access by secreting and ensheathing extracellular matrix-based conduit networks that rapidly convey small, soluble lymph-borne molecules to the SLO core. Furthermore, lymph node stromal cells (LNSCs) directly induce \(CD8^+\) T cell tolerance to peripheral tissue restricted antigens and constrain the proliferation of newly activated T cells in these sites. Thus, stromal-hematopoietic interactions are crucial for the normal functioning of the immune system. LNSCs are extremely rare and difficult to isolate, hampering the thorough study of their biology. In order to better understand these stromal subsets, we sorted fibroblastic reticular cells (FRCs), lymphatic endothelial cells, blood endothelial cells, and podoplanin \(^−CD31^−\) cells (double negative stromal cells; DNCs) to high purity from resting and inflamed murine lymph nodes. We meticulously analyzed the transcriptional profiles of these freshly isolated LNSCs as part of the Immunological Genome Project Consortium. Analysis of the transcriptional profiles of these LNSC subsets indicated that SCs express key immune mediators and growth factors, and provided important insights into the lymph node conduit network, FRC-specialization, and the DNC identity. Examination of hematopoietic and stromal transcription of ligands and cognate receptors suggested complex crosstalk among these populations. Interestingly, FRCs dominated cytokine and chemokine transcription among LNSCs, and were also enriched for higher expression of these genes when compared with skin and thymic fibroblasts, consistent with FRC-specialization. LNSCs that were isolated from inflamed lymph nodes robustly upregulated expression of genes encoding cytokines, chemokines, antigen-processing and presentation machinery, and acute-phase response molecules. Little-explored DNCs showed many transcriptional similarities to FRCs, but importantly did not transcribe interleukin-7. We identified DNCs as consisting largely of myofibroblastic pericytes that express integrin \(\alpha 7\). Together these data comprehensively describe the transcriptional characteristics of four major LNSC subsets isolated from resting and inflamed SLOs, offering many avenues for future study.