Nascent mesodermal cells derived from EB5 embryonic stem (ES) cells were sorted in terms of cardiogenic potential on the basis of their expression levels of platelet-derived growth factor receptor alpha (PDGFR alpha) and fetal liver kinase 1 (Flk-1). The sorted cells were cocultured with OP9 stromal cells to induce terminal differentiation into contractile cardiac colonies. A significant number of cardiac colonies were found in the Flk-1+/PDGFR alpha+ fraction. The enrichment double-positive fraction produced approximately fivefold more cardiac colonies than the Flk-1+/PDGFR alpha-fraction and 10-fold more than the Flk-1-/PDGFR alpha+ fraction. To investigate the involvement of these markers in embryonic cardiogenesis, the cells that disseminated from the E7.5-7.75 embryos were fractionated and seeded on OP9 cells. The cardiogenic potential was markedly enhanced in the Flk-1+/PDGFRa+ fraction. These results suggest that some of the precursor cells coexpressing these markers are selectively involved in cardiogenic events, and that the identification of ES-cell-derived precursors with these markers will contribute to the effective production of cardiomyocytes for cell therapies.