Spermatogonial stem cells (SSCs) reside within an ‘open niche’ in the testis that emanates key regulatory signals to balance self-renewal versus differentiation. It has been proposed that vicinity to testicular vasculature (and thus oxygen availability) may influence SSC fate, as is the case multiple stem cell niches. In support of this, our analysis of single cell RNA-sequencing (scRNA-seq) data produced from postnatal day 6 mouse spermatogonia revealed enrichment of hypoxia-inducible transcription factors (HIFs) in the SSC population (Gfra1+, Id4+). Contrastingly, normoxia-responsive factors that tag HIF proteins for degradation were enriched in progenitor (Neurog3+, Sox3+) and differentiating (Stra8+, Kit+) spermatogonia. We elected to further characterise HIF2A (“EPAS1”) expression using the Id4-eGfp transgenic mouse to identify SSCs (GFP-‘Bright’ cells). Using immunofluorescence techniques, over 90% of SSCs were found to express EPAS1, compared to <10% of progenitor spermatogonia (P<0.001). Similarly, when Id4-eGfp mice were injected with ‘hypoxyprobe’ (pimonidazole), hypoxia-associated protein adducts were identified in >80% SSCs compared to <10% progenitor spermatogonia (P<0.001). To determine a functional role for EPAS1 in SSC regulation, a germline-specific Epas1-knockout mouse line was produced (“Epas1-cKO”). Epas1-cKO males are fertile up to 6 months of age (breeding study ongoing), however quantitative differences in spermatogenesis are apparent. Notably, at 6 months, Epas1-cKO testis weight is increased by 10%, epididymis weight by 30%, and caudal sperm concentration by 10%, when compared to control animals (n=1). Contrastingly, sperm motility and viability are decreased by 65% and 40%, respectively. Quantitative differences in sperm production in Epas1-cKO mice may suggest perturbation of mitosis in slow-cycling SSCs, or a shift in the SSC-progenitor equilibrium: possibilities that are currently under investigation. These data suggest that at least a subset of SSCs preferentially reside in hypoxic microenvironments in the testis, and that HIFs may influence cell fate decisions and/or mitotic regulation in these cells.