Mammalian spermatogenesis is maintained by stem cell capacity within undifferentiated spermatogonial subpopulation. Here, using a combination of surface markers, we describe a purification method for undifferentiated spermatogonia. Flow cytometric analysis revealed that this population is composed of Plzf-positive cells and exhibits quiescence and the side population phenotype, fulfilling general stem cell criteria. We then applied this method to analyze undifferentiated spermatogonia and stem cell activity of Atm −/− mice. Atm −/− testis shows 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 of the p19 Arf -p53-p21 Cip1/Waf1 pathway were observed in Atm −/− undifferentiated spermatogonia. Moreover, suppression of p21 Cip1/Waf1 in an Atm −/− background restored transplantation ability of undifferentiated spermatogonia, indicating that ATM plays an essential role in maintenance of undifferentiated spermatogonia and their stem cell capacity by suppressing DNA damage-induced cell-cycle arrest.