Male sex determination hinges on the development of testes in the embryo, beginning with the differentiation of Sertoli cells under the influence of the Y-linked gene SRY. Sertoli cells then orchestrate fetal testis formation including the specification of fetal Leydig cells (FLCs) that produce steroid hormones to direct virilization of the XY embryo. As the majority of XY disorders of sex development (DSDs) remain unexplained at the molecular genetic level, we reasoned that genes involved in FLC development might represent an unappreciated source of candidate XY DSD genes. To identify these genes, and to gain a more detailed understanding of the regulatory networks underpinning the specification and differentiation of the FLC population, we developed methods for isolating fetal Sertoli, Leydig, and interstitial cell-enriched subpopulations using an Sf1-eGFP transgenic mouse line. RNA sequencing followed by rigorous bioinformatic filtering identified 84 genes upregulated in FLCs, 704 genes upregulated in nonsteroidogenic interstitial cells, and 1217 genes upregulated in the Sertoli cells at 12.5 days postcoitum. The analysis revealed a trend for expression of components of neuroactive ligand interactions in FLCs and Sertoli cells and identified factors potentially involved in signaling between the Sertoli cells, FLCs, and interstitial cells. We identified 61 genes that were not known previously to be involved in specification or differentiation of FLCs. This dataset provides a platform for exploring the biology of FLCs and understanding the role of these cells in testicular development. In addition, it provides a basis for targeted studies designed to identify causes of idiopathic XY DSD.