Skeletal muscle differentiation is mediated by a complex gene expression program requiring both the muscle-specific transcription factor Myogenin (Myog) and p38α MAPK (p38α) signaling. However, the relative contribution of Myog and p38α to the formation of mature myotubes remains unknown. Here, we have uncoupled the activity of Myog from that of p38α to gain insight into the individual roles of these proteins in myogenesis. Comparative expression profiling confirmed that Myog activates the expression of genes involved in muscle function. Furthermore, we found that in the absence of p38α signaling, Myog expression leads to the down-regulation of genes involved in cell cycle progression. Consistent with this, the expression of Myog is sufficient to induce cell cycle exit. Interestingly, p38α-defective, Myog-expressing myoblasts fail to form multinucleated myotubes, suggesting an important role for p38α in cell fusion. Through the analysis of p38α up-regulated genes, the tetraspanin CD53 was identified as a candidate fusion protein, a role confirmed both ex vivo in primary myoblasts, and in vivo during myofiber regeneration in mice. Thus, our study has revealed an unexpected role for Myog in mediating cell cycle exit and has identified an essential role for p38α in cell fusion through the up-regulation of CD53.