Differences of Sex Development are congenital conditions in which development of chromosomal, gonadal, or anatomical sex is atypical. Options for functional analysis of variants identified in patients is limited to in vitro cell models, biochemical assays and mouse models. Often, the functional consequences of causative variations cannot be elucidated using these methods as there is no cell model that perfectly mimics gonadal cells and there is many key differences between mouse and human gonadal development. We have tried to mitigate this limitation by reprogramming readily available skin tissue derived dermal fibroblasts into Sertoli cells (SC), which could then be used in function assays. We employed a computational predictive algorithm for cell conversions called Mogrify to predict the transcription factors (TFs) required for direct reprogramming of human dermal fibroblasts into SCs. We established trans-differentiation culture conditions where stable transgenic expression of these TFs was achieved in 46, XY adult dermal fibroblasts using lentiviral vectors. The resulting Sertoli like cells (SLCs) were validated for SC phenotype using several approaches. These cells exhibited Sertoli like morphological and biophysical properties that differed as revealed by morphometry and xCELLigence assays. They also showed Sertoli-specific expression of molecular markers such as SOX9, PTGDS, BMP4, or DMRT1 as revealed by IF imaging, RNAseq and qPCR. These cells additionally lacked expression of markers of other gonadal cell types such as Leydig, germ, peritubular myoid or granulosa cells. The trans-differentiation method was also applied to a commercially available 46, XY fibroblast line derived from a patient with DSD. The resulting cells displayed lower levels of SOX9 (a key testis marker) in comparison to normal 46, XY derived SLCs and also showed impaired proliferation in an xCELLigence assay, thus showcasing the robustness of this new trans-differentiation model.