Asingle cells in undifferentiated spermatogonia are considered to be the most primitive forms of germ stem cells (GSCs). Although GFRα1 is thought to be a marker of Asingle cells, we found that Bmi1(High) is more specific than GFRα1 for Asingle cells. Bmi1(High) expression in Asingle cells is correlated with seminiferous stages, and its expression was followed by the proliferative stage of Asingle GSCs. In contrast, GFRα1 expression was seminiferous stage-independent. Fate analyses of EdU-positive Bmi1(High)-positive cell-derived Asingle cells revealed that these cells self-renewed or generated transient amplifying Apaired cells. Bmi1(High)-positive cells were resistant to irradiation-induced injury, after which they regenerated. Elimination of Bmi1(High)-positive cells from seminiferous tubules resulted in the appearance of tubules with seminiferous stage mismatches. Thus, in this study, we found that Bmi1(High) is a seminiferous stage-dependent marker for long-term GSCs and that Bmi1(High)-positive cells play important roles in maintaining GSCs and in regenerating spermatogenic progenitors after injury.