Differences of Sex Development (DSD) are a spectrum of conditions in which gonadal, chromosomal or anatomical sex is atypical. The underlying molecular basis remains unknown in many forms of DSD, due in part to the lack of manipulable models that recapitulate human sex determination. Mouse models have been useful in studying the function of critical sex determination genes using knockout/transgenic approaches, yet there are limitations when exclusively relying on these models. Functional redundancy, gene dosage or genetic buffering (alternative pathways existing for the same functional outcome) often result in no phenotypic consequence. Furthermore, differences of gene expression thresholds and genetic robustness between humans and mice are becoming apparent. An in vitro model that can be used to model Sertoli cell function in the testis is NT2/D1, a human pluripotent clonal cell line derived from a testicular tumour. Expressing the testis-determining genes SRY, SOX9, DHH and FGF9, with an absence of ovary-specific FOXL2 and WNT4, NT2/D1 cells can be used in their undifferentiated state as a model for human Sertoli cell function. We have established an NT2/D1-cas9 cell line, and characterised these via a suite of cell phenotyping assays including xCELLigence® RTCA, assessing cellular behaviours analogous to Sertoli Cell behaviours and functions. This model can be used to investigate multiple SOX9 target genes, to assess their individual contributions to cell adhesion, proliferation and polarity – all important behaviours of Sertoli cells to direct testicular architecture in the developing male gonad. There is a need for high-throughput evaluation of candidate DSD genes and variants; unknown DSD genes may be SOX9 target genes, which need further characterisation. Cas9 engineered deletion and missense mutations from DSD patients will help to pinpoint the processes that differ in cells in XY-female versus typical XY-males.