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 animal models. Often, the functional consequences of causative variations cannot be elucidated using these methods as there is no cell model that authentically mimics fetal 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. SLC shape and size resemble mature human Sertoli cells, as were cell adhesion and proliferation. RNAseq revealed Sertoli-cell specific genes such as SOX9, PTGDS, BMP4, or DMRT1 were expressed as validated by IF. Conversely, 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 a case of testicular dysgenesis of unknown (exome-negative) genetic etiology, the intersex SLCs express low levels of SOX9 consistent with the clinical diagnosis. This model will aid in variant interpretation following WGS towards improved diagnosis and clinical management of intersex.