Mammalian spermatogenesis is a classic adult stems cell-dependent process, supported by the self-renewal and differentiation of spermatogonial stem cells (SSCs). However, the identification of SSCs and elucidation of their behaviors in undisturbed testis has long been a big challenge. Here, we generated a knock-in mouse model, Id4-2A-CreERT2-2A-tdTomato, which allowed us to mark Id4-expressing (Id4(+)) cells at different time points in situ and track their behaviors across distinct developmental stages during steady-state and regenerating spermatogenesis. We found that Id4(+) cells continue to produce spermatogonia, spermatocytes and sperm in mouse testis, showing they are capable of self-renewal and have differentiation potential. Consistent with these findings, ablation of Id4(+) cells in mice results in a loss of spermatogenesis. Furthermore, developmental fate mapping reveals that Id4(+) SSCs originate from neonate Id4(+) gonocytes. Therefore, our results indicate that Id4 marks spermatogonial stem cells in the mouse testis.