Despite the ability of myogenic progenitor cells (MPCs) to completely regenerate skeletal muscle following injury, little is known regarding the molecular program that regulates their proliferation and differentiation. Although mice lacking the cyclin dependent kinase inhibitor, p21 (p21-/-), develop normally, we report here that p21-/- MPCs display increased cell number and enhanced cell cycle progression compared to wildtype MPCs. Therefore, we hypothesized that p21-/- mice would demonstrate temporally-enhanced regeneration following myotrauma. In response to cardiotoxin-induced injury, p21-/- skeletal muscle regeneration was significantly attenuated versus regenerating wildtype muscle, contrary to the hypothesis. Regenerating p21-/- skeletal muscle displayed increased proliferative (PCNA positive) nuclei coincident with increased apoptotic nuclei (TUNEL positive) compared to wildtype muscle up to 3 weeks following injury. Differentiation of p21-/- MPCs was markedly impaired and associated with increased apoptosis compared to wildtype MPCs confirming that the impaired differentiation of the p21-/- MPCs was a cell autonomous event. No dysregulation of p27, p53 or p57 protein expression in differentiating p21-/- MPCs compared to wildtype MPCs was observed suggesting that other compensatory mechanisms are responsible for the regeneration that ultimately occurs. Based on these findings, we propose that p21 is essential for the coordination of cell cycle exit and differentiation in the adult MPC population and in the absence of p21, skeletal muscle regeneration is markedly impaired.