In mammals, male fate is under the control of the master transcriptional regulator, SOX9: in its presence, somatic precursor cells of the embryonic gonads differentiate into Sertoli cells, the central organizers of testicular differentiation. Therefore, analyzing target genes of this transcription factor allows the elucidation of cellular commitment mechanisms at the genome level. With the use of ChIP-seq in murine and bovine wild-type testes combined with RNAseq from mouse testes lacking SOX9, we identified SOX9 target genes in the mammalian fetal gonad. SOX9 in fetal testes binds to a large set of genes conserved among mammals, including those with well-established roles in testis and ovary development. RNAseq analysis shows that testes and ovaries display sex-specific RNA splicing and that SOX9 mediates both target transcription and differential splicing of these target genes. Regions bound by SOX9 are predominantly 5′ proximal or intra-genic, and display specific genomic features we call "Sertoli Cell Signatures" or SCS. The SCS is conserved among mammals and comprises multiple binding motifs for the Sertoli reprogramming factors SOX9, GATA4 and DMRT1; indeed, independent DMRT1 ChIP-seq confirms the enrichment of the SCS. Bioinformatic analysis of SCS regions predicts novel regulatory mechanisms prompting functional validation, and examples will be discussed. Also, target genes are also being validated by in vivo (XY KO or XX KI mice) and ex vivo (drug treatments of cultured gonads) approaches which will be described. This work is unravelling the regulatory networks during gonadal development and identifying novel sex determining genes, whose functions could be altered in patients with Disorders of Sex development, most of whose forms remain unexplained genetically.