Mammalian spermatogenesis is maintained by stemPDE signaling cell capacity inside undifferentiated spermatogonial subpopulation. Here, using a blend of surface markers, we describe a purification system for undifferentiated spermatogonia. Flow cytometric examination unveiled that this population is composed of Plzf-positive cells and exhibits Interleukin-2 receptor quiescence and the side population phenotype, fulfilling common stem cell criteria. We then utilized this process to analyze undifferentiated spermatogonia and stem cell exercise of Atm(-/-) mice. Atm(-/-) testis exhibits progressive depletion of undifferentiated spermatogonia accompanied by cell-cycle arrest. In Atm(-/-) undifferentiated spermatogonia, a self-renewal defect was observed in vitro and in vivo. Accumulation of DNA damage and activation in the p19(Arf)-p53-p21(Cip1/Waf1) pathway had been observed in Atm(-/-) undifferentiated spermatogonia. Also, suppression of p21(Cip1/Waf1) in an Atm(-/-) background restored transplantation potential of undifferentiated spermatogonia, indicating that ATM plays an critical position in servicing of undifferentiated spermatogonia and their stem cell capacity by suppressing Tenatoprazole DNA damage-induced cell-cycle arrest.