In mammalian testes, “A-single” spermatogonia function as stem cells that sustain sperm production for fertilizing eggs. Yet, it is not understood how cellular niches regulate the developmental fate of A-single spermatogonia. Here, immunolabeling studies in rat testes define a novel population of ERBB3 germ cells as approximately 5% of total SNAP91 A-single spermatogonia along a spermatogenic wave. As a function of time, ERBB3 A-single spermatogonia are detected during a 1- to 2-day period each 12.9-day sperm cycle, representing 35%–40% of SNAP91 A-single spermatogonia in stages VIII–IX of the seminiferous epithelium. Local concentrations of ERBB3 A-single spermatogonia are maintained under the mean density measured for neighboring SNAP91 A-single spermatogonia, potentially indicative of niche saturation. ERBB3 spermatogonia also synchronize their cell cycles with epithelium stages VIII–IX, where they form physical associations with preleptotene spermatocytes transiting the blood-testis barrier and Sertoli cells undergoing sperm release. Thus, A-single spermatogonia heterogeneity within this short-lived and reoccurring microenvironment invokes novel theories on how cellular niches integrate with testicular physiology to orchestrate sperm development in mammals.