Embryonic stem (ES) cell-derived cardiomyocytes (ESCMs) must be specifically purified in order to prevent teratoma formation, and this confusing issue has hampered their clinical application. We therefore investigated a technique to generate pure labeled ESCMs for possible use in cardiac repair. We generated transgenic ES cell lines expressing enhanced green fluorescent protein (EGFP) under the transcriptional control of the alpha-cardiac myosin heavy chain (alpha-MHC) promoter. Differentiated EGFP-positive ES cells displayed characteristics of CMs. Furthermore, neuregulin-1 (NRG-1) upregulated the expression of the cardiac-restricted transcription factors Nkx2.5 and GATA-4, as well as differentiated CM factors (alpha-MHC, beta-MHC). Immunohistochemistry demonstrated that NRG-1 increased expression of cardiac-specific troponin T in the beating foci of the embryoid bodies. This work revealed a potential method for specifically labeling and enriching ESCMs by combining genetically-engineered ES cell clones and exogenous growth factor treatment.