Sex determination refers to the decision of the bipotential early gonads to develop as either testes or ovaries during embryogenesis. In mammals, a single genetic trigger involved in this pivotal decision has been identified on the Y chromosome: the testis-determining gene SRY/Sry. During embryogenesis, SRY triggers the differentiation of Sertoli cells from the supporting cell precursor lineage which would otherwise give granulosa cells in ovaries. Several testis-specific events occur after SRY expression and the onset of Sertoli cell differentiation, notably Leydig cell differentiation, testis cord formation, and development of testis-specific vasculature. Although a number of genes involved in these events have been identified, how they relate to Sry action is poorly understood. Furthermore, even at the adult stage, some of these genes retain a key role in maintaining the testicular fate because conditional ablation of the genes leads to adult testis dysgenesis or transdifferentiation into an ovary. This sheds light on mammalian sex-reprogramming, despite the prevailing dogma that postnatal sex change does not occur in mammals. In this review, we summarize our current understanding of genetic pathways of testis determination and differentiation in mammals, particularly in the mouse and the human.