Alternative splicing contributes largely to cell differentiation and functional specification. We previously reported that the RNA-binding protein RBM4 antagonizes the activity of splicing factor PTB to modulate muscle cell–specific exon selection of α-tropomyosin. Here we show that down-regulation of PTB and its neuronal analogue nPTB during muscle cell differentiation may involve alternative splicing-coupled nonsense-mediated mRNA decay. RBM4 regulates PTB/nPTB expression by activating exon skipping of their transcripts during myogenesis. Moreover, RBM4 and PTB target a common set of transcripts that undergo muscle cell–specific alternative splicing. Overexpression of RBM4 invariably promoted expression of muscle cell–specific isoforms, which recapitulated in vivo alternative splicing changes during muscle differentiation, whereas PTB acted oppositely to RBM4 in expression of mRNA isoforms specific for late-stage differentiation. Therefore, RBM4 may synergize its effect on muscle cell–specific alternative splicing by down-regulating PTB expression and antagonizing the activity of PTB in exon selection, which highlights a hierarchical role for RBM4 in a splicing cascade that regulates myogenesis.