Although embryonic stem (ES) cells (ESCs) may be a promising donor source for the repair of infarcted or ischemic heart tissues, their successful application in regenerative medicine has been hampered by difficulties in enriching, identifying, and selecting cardiomyocytes from the differentiating cells. We established transgenic human ES cell lines by transcriptional control of the alpha-cardiac myosin heavy chain (alpha-MHC) promoter driving green fluorescent protein (GFP) expression. Differentiated GFP-expressing cells display the characteristics of cardiomyocytes (CMs). Apela, a recently identified short peptide, up-regulated the expression of the cardiac-restricted transcription factors Tbx5 and GATA4 as well as differentiated the cardiomyocyte markers alpha-MHC and beta-MHC. Flow cytometric analysis showed that apela increased the percentage of GFP-expressing cells in the beating foci of the embryoid bodies. The percentage of cardiac troponin T (TNT)-positive cells and the protein expression of TNT were increased in the ES cell-derived CMs with apela treatment. Functionally, the contractile frequency of the ES-derived CMs responded appropriately to the vasoactive drugs isoprenaline and carbachol. Our work presented a protocol for specially labelling and enriching CMs by combining transgenic human ES cell lines and exogenous growth factor treatment.