Spermatogonial stem cells (SSCs) are essential for male fertility. How they initially form is poorly understood. Here, we report that SSC generation is driven by a transcription factor (TF) cascade controlled by the homeobox protein RHOX10, which acts by stimulating the differentiation of SSC precursors called pro-spermatogonia (ProSG). We identified genes regulated by RHOX10 in ProSG in vivo using genome-wide transcriptome profiling. We defined RHOX10-direct target genes using the CUT&Tag protocol coupled with a rapid temporal induction assay we designed: inducible thiol(SH)-linked alkylation for the metabolic sequencing of RNA (iSLAMseq). Together, these genome-wide assays identified temporal waves of RHOX10-direct targets, as well as secondary-target genes. Many of these RHOX10-regulated genes encode proteins with known roles in SSCs. Using an in vitro ProSG differentiation assay that we developed, we found that RHOX10 promotes ProSG differentiation through a conserved transcriptional cascade involving the key germ-cell TFs DMRT1 and ZBTB16. Our study gives important insights into germ cell development and provides a blueprint for how to define TF cascades.