Dissection of cardiomyocyte differentiation process at the cellular level is indispensable in the research for cardiac development and regeneration. Previously, we have established an embryonic stem cell differentiation system that reproduces early vascular development from progenitor cells that express Flk1, a vascular endothelial growth factor receptor, by the combinatory application of 2-dimensional culture and flowcytometry. Here we show that cardiomyocytes can be successfully induced from a single Flk1+ cell on 2-dimensional culture, enabling the direct observation of differentiating cardiomyocytes and the prospective identification of cardiac progenitor potentials. Flk1+ cells could give rise to cardiomyocytes, as well as endothelial cells, from a single cell by the co-culture on OP9 stroma cells in a fusion-independent manner. Among the cell populations in intermediate stages from Flk1+ cells to cardiomyocytes, Flk1+/CXCR4+/vascular endothelial cadherin- cells were cardiac-specific progenitors at the single cell level. Noggin, a bone morphogenetic protein inhibitor, abolished cardiomyocyte differentiation by inhibiting the cardiac progenitor induction. However, wnt inhibitors Dkk-1 or Frizzled-8/Fc chimeric protein augmented, but wnt3a inhibited, cardiomyocyte differentiation. In vitro reproduction of cardiomyocyte differentiation process should be a potent tool for the cellular and molecular elucidation of cardiac development, which would provide various targets for cardiac regeneration.