SOX9, a DNA binding transcriptional activator, is the main regulator of mammalian testis development and plays a major role in Sertoli cell differentiation, testis development and fertility. Target genes of SOX9 have previously been characterised such as AMH, FGF9 and PTGDS but due to the high number of idiopathic 46,XY DSD cases, we suspect that there are more. Here, we utilise transcriptome analysis of E13.5 Sox9 knock-out gonads in order to identify SOX9-responsive genes in the developing testis. The candidate genes include DHH, ETV5 and NEDD9. To investigate the mechanisms of regulation of these genes, an in vitro approach was utilized. Candidate genes were validated in NT2/D1 cells by assessing their response to SOX9 overexpression and knockdown. In silico analysis of DHH and ETV5 promotor regions was used to assess the binding potential of SOX9 and sites were validated using SOX9 ChIP-seq in NT2/D1 cells, luciferase assay and EMSA. Immunohistochemistry was used to visualise the localisation of NEDD9 and ETV5 in E13.5 mouse testis in Sertoli cells. SOX9 ChIP-seq in embryonic bovine gonads was also analysed.NT2/D1 cells transiently over-expressing SOX9 or knockdown with siRNA reveals that DHH, ETV5 and NEDD9 respond significantly in the manner to which the SOX9 expression is altered. In silico analysis of DHH and ETV5 promotor regions revealed potential binding sites which were confirmed by ChIP-seq analysis of NT2/D1 cells, luciferase assay and EMSA. A peak in the bovine SOX9 ChIP-seq across the promoter region of NEDD9 also reveals a potential binding site for future analysis. Immunolocalisation of NEDD9 in the mouse testis revealed its expression in the Sertoli cell cytoplasm and ETV5 in the nucleus at E13.5.DHH, ETV5 and NEDD9 are all likely direct targets of SOX9 in the developing testis. Further analysis of NEDD9 is planned including the assessment of knockout gonads.