Spermatogonial stem cells (SSCs) are essential for the generation of sperm, a developmental process that occurs continuously in sexual mature males. SSCs are important clinically, as SSC defects are implicated in many cases of male infertility. In addition, there is an interest in using SSCs to cure patients with defects in later stages of spermatogenesis. While some progress has been made in understanding some of the fundamental characteristics of SSCs, how SSCs are initially generated and maintained in vivo remains poorly understood. We have identified a transcription factor, RHOX10, that our studies have suggested has roles in SSCs. When the Rhox10 gene is mutated in mice, this causes phenotypic defects consistent with a dramatic loss in SSCs. During the progress period, we used this mouse mutant model coupled with a battery of approaches to examine whether indeed RHOX10 is required for the normal accumulation of SSCs. Using a germ cell transplantation assay, which is regarded as the gold standard for assaying SSC numbers, we found that Rhox10-knockout (KO) mice have dramatically lower numbers of SSCs than wild-type control mice. This SSC defect was observed at an early postnatal age, raising the possibility that RHOX10 is important for the initial establishment of SSCs, a developmental step that occurs soon after birth in mice. In further support of a dramatic reduction in SSCs, we found that both postnatal and adult Rhox10-KO mice have reduced numbers of testicular cells expressing markers known to be expressed on SSCs. In principal, the reduction in SSCs could be caused by an embryonic defect, such as in the formation or expansion of primordial germ cells in the embryo. We found that this is unlikely to be the case, as we observed normal numbers of germ cells in the testis of Rhox10-KO mice at late points of embryonic development and at birth. We then took a series of approaches to assess how Rhox10 functions to drive the initial establishment of SSCs, a developmental step that occurs postnatally. Assessment of proliferation and apoptosis markers demonstrated that there was no measurable defect in the proliferation or survival of SSCs or the germ cells that give rise to them: pro-spermatogonia (Pro-SG). After ruling out these and other possibilities, we considered that Rhox10 acts instead by promoting the differentiation of Pro-SG into SSCs. To test this, we took advantage of markers relatively specific for Pro-SG and SSCs, coupled with reporter mice and double-immunofluorescence analysis. Together, the data we obtained provided strong support for the notion that, indeed, Rhox10 is necessary for the efficient conversion of Pro-SG into SSCs. To explore this in more depth, we used a new genome wide approach – single cell-RNAseq analysis. Comparison of single germ cells from early postnatal Rhox10-KO and control mice confirmed that loss of Rhox10 causes a defect in the progression of Pro-SG into SSCs. Single cell-RNAseq analysis also identified putative Rhox10 target genes in specific germ cell subsets, including in Pro-SG and SSCs. Finally, this approach identified many transcripts highly enriched in specific wild-type and Rhox10-null germ cell subsets that are candidates to serve as Pro-SG- and SSC-specific markers. This is a critical advancement for the field, as currently no such markers exist. Given that Pro-SG are considered to be the immediate precursor cells of SSCs, having markers that define them will be broadly useful for future studies on this critical developmental step. SSC-specific markers will have major utility for both basic science research and for future clinical applications of SSCs. In conclusion, during the last progress period, we obtained definitive evidence that Rhox10 is critical for the initial generation of mouse SSCs. We obtained a wealth of evidence that RHOX10 promotes the differentiation of SSC precursor cells (Pro-SG) into SSCs. To our knowledge, RHOX10 is the first factor shown to have a role in this developmental step. Given that Pro-SG are a transient and finite population of germ cells that are considered to be the soul source of SSCs, this is an important advance. Another goal of our study was to determine whether RHOXF2, a candidate human ortholog of mouse Rhox10, also functions in SSCs. Towards this goal, during the progress period, we optimized culture and lentivirus infection conditions for germ cells obtained from human testicular biopsies. We also initiated experiments to knockdown RHOXF2 in these cultures using RNA interference.