Upon encounter of an antigen, epidermis-resident Langerhans cells (LCs) become activated and present the processed antigen to T cells of the draining lymph nodes, resulting in tolerogenic or inflammatory responses. In psoriasis plaques, skin homeostasis is disrupted and replaced by an inflammatory dermatitis. Topical application of the anti-viral compound, imiquimod, induces a psoriasiform inflammatory condition, partly driven by LC production of pro-inflammatory cytokines. Differentiation of the myeloid progenitor cell line, MUTZ-3, produces MUTZ-3 derived Langerhans cells (MUTZ-LCs) which can be used as an in vitro model of LCs. This project aimed to investigate the phenotype and function of imiquimod-treated MUTZ-LCs in monolayer cultures, co-culture with T cells and inserted into a 3D skin model. LC-related surface markers (HLA-DR, CD1a, CD207, CCR7) were upregulated in MUTZ-LCs after 7 days of differentiation with 40 ng/ml GM-CSF, 10 ng/ml TGF-β and 2.5 ng/ml TNF-α. Supernatants of imiquimod-treated monolayer cultures of MUTZ-LCs showed subtle concentrations of IL-6 and TNF-α, but not IL-23. mRNA expression showed no significant upregulation of IL-6, IL-23 or TNF-α after 24 h treatment with imiquimod. The presence of MUTZ-LCs in T cell co-cultures greatly increased the production of IL-2, but did not affect expression of CD25. After 16 h exposure to imiquimod, IL-6, IL-23 and TNF-α could not be detected in culture supernatants of a 3D model consisting of fibroblasts, keratinocytes and MUTZ-LCs. The model was devoid of fibroblasts after 19 days of culture, most likely compromising the immunocompetence, as LC migration in response to activation could not be detected. Further studies could refine and optimize the imiquimod-3D skin model, which has potential as a possible substitute for animal models in psoriasis research.