Extensive analyses of mice carrying null mutations in paired box 7 (Pax7) have confirmed the progressive loss of the satellite cell lineage in skeletal muscle, resulting in severe muscle atrophy and death. A recent study using floxed alleles and tamoxifen-induced inactivation concluded that after 3 wk of age, Pax7 was entirely dispensable for satellite cell function. Here, we demonstrate that Pax7 is an absolute requirement for satellite cell function in adult skeletal muscle. Following Pax7 deletion, satellite cells and myoblasts exhibit cell-cycle arrest and dysregulation of myogenic regulatory factors. Maintenance of Pax7 deletion through continuous tamoxifen administration prevented regrowth of Pax7-expressing satellite cells and a profound muscle regeneration deficit that resembles the phenotype of skeletal muscle following genetically engineered ablation of satellite cells. Therefore, we conclude that Pax7 is essential for regulating the expansion and differentiation of satellite cells during both neonatal and adult myogenesis.