Transcription factors of the basic helix-loop-helix (bHLH) family are critical regulators of muscle cell differentiation. For example, Myod drives skeletal muscle differentiation, and Hand2 potentiates cardiac muscle differentiation. Understanding how these bHLH factors regulate distinct transcriptional targets in a temporally and spatially controlled manner is critical for understanding their activity in cellular differentiation. We previously showed that Pbx homeodomain proteins modulate the activity of Myod to promote the differentiation of fast-twitch skeletal muscle. Here, we test the hypothesis that Pbx proteins are also necessary for cardiac muscle differentiation through interacting with Hand2. We show that Pbx proteins are required for the activation of cardiac muscle differentiation in zebrafish embryos. Loss of Pbx activity leads to delay of myocardial differentiation and subsequent defective cardiac morphogenesis, similar to reduced Hand2 activity. Genetic interaction experiments support the hypothesis that Pbx proteins modulate the activity of Hand2 in myocardial differentiation. Furthermore, we show that Pbx proteins directly bind the promoter of the myocardial differentiation gene myl7 in vitro, supporting a direct role for Pbx proteins in promoting cardiac muscle differentiation. Our findings demonstrate new roles for Pbx proteins in vertebrate cardiac development and also provide new insight into connections between the transcriptional regulation of skeletal and cardiac muscle differentiation programs.