Pax3 is an essential myogenic regulator of fetal and embryonic development, but its role in postnatal myogenesis remains a topic of debate. We show that constitutive expression of Pax3 in postnatal, juvenile mouse skeletal muscle stem cells, a subset of the heterogeneous satellite cell pool highly enriched for myogenic activity, potently induces differentiation. This differentiation-promoting activity stands in contrast to the differentiation-inhibiting effects of Pax3 in the commonly used mouse myoblast cell line C2C12. Pax3 mRNA levels in distinct muscles correlate with the rate of myogenic differentiation of their muscle stem cells. Although Pax3 controls embryonic myogenesis through regulation of the canonical myogenic regulatory factors (MRFs) Myf-5, MyoD, myogenin and Mrf4, we find that in postnatal muscle stem cells, ectopic Pax3 expression fails to induce expression of any of these factors. Unexpectedly, overexpression of neither Myf-5 nor myogenin is sufficient to induce differentiation of juvenile stem cells; and knockdown of Myf-5, rather than inhibiting differentiation, promotes it. Taken together, our results suggest that there are distinct myogenic regulatory pathways that control the embryonic development, juvenile myogenesis and adult regeneration of skeletal myofibers.