Signaling from the retinoic acid receptors (RARs) and retinoid X receptors (RXRs) is essential for cardiovascular morphogenesis in vivo. RAR and/or RXR signaling can also enhance the in vitro induction of cardiomyocytes from murine embryonic stem (ES) cells in the presence of serum. The present study examined the effect of RXR agonist that was specifically bound to RXRs on the differentiation of mouse ES cells into cardiomyocytes in vitro in the absence of serum. The number of beating embryoid body-like spheres (EBSs) derived from the ES cells increased significantly following treatment with PA024, an RXR agonist. In contrast, when EBSs were treated with PA452, which was specifically bound to RXR and worked as an antagonist, the number of beating EBSs was decreased in a dose-dependent manner. These results suggest that RXR signaling regulates cardiomyocyte numbers during the differentiation of ES cells in vitro and probably in normal development.