Intersex are congenital conditions in which development of chromosomal, gonadal, or anatomical sex is atypical. After WGS, options for functional analysis of causative DNA variants identified in patients is limited to 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 authentically mimics gonadal somatic cell types in which variants are expressed development. We have tried to mitigate this limitation by reprogramming readily available skin tissue derived dermal fibroblasts into Sertoli cells, which could then be used in functional 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 Sertoli cells. We established trans-differentiation culture conditions where stable expression of TFs was achieved in 46, XY adult dermal fibroblasts using lentiviral vectors. The resulting Sertoli-like cells (SLCs) were validated for Sertoli cells characteristics by several approaches. Transdifferentiation into SLCs led to shape and size changes to those resembling human Sertoli cells. Cell proliferation changes (xCELLigence assays) were typical of Sertoli cells nearing maturation. Also, Sertoli-cell specific genes such as SOX9, PTGDS, BMP4, or DMRT1 were expressed, while markers of other testicular cell types and of the ovary were by and large not expressed. The trans-differentiation method was also applied to four fibroblast lines from 46, XY intersex patients. In one case of testicular dysgenesis of unknown (exome-negative) genetic etiology, the SLCs expressed lower levels of SOX9 consistent with the clinical diagnosis. This model will aid in variant interpretation following WGS towards improved diagnosis of intersex and clinical management.