Inefficient cardiomyocyte differentiation limits the therapeutic use of embryonic stem (ES) cell-derived cardiomyocytes. While large collections of proprietary chemicals had been screened to improve ES cell differentiation into cardiomyocytes, the natural product library remained unexplored. Using a mouse ES cell line transfected with a cardiomyocyte-specific alpha-myosin heavy chain promoter-driven enhanced green fluorescent protein (EGFP) reporter, we screened 24 natural products with known cardioprotective actions. Salvianolic acid B (saB), while produced minimal effect on its own, concentration-dependently synergized with vitamin C in inducing cardiomyocyte differentiation, as demonstrated by an increase in EGFP(+) cells, beating area in embryoid bodies, and expression of cardiomyocyte maturity markers. This synergy is specific to cardiomyocyte differentiation, and is involved with collagen synthesis. The present study demonstrates the saB-vitamin C synergy in inducing ES cell differentiation into matured and functional cardiomyocytes, and this may lead to a practicable cocktail approach to generate ES cell-derived cardiomyocytes for cardiac stem cell therapy. (C) 2009 Elsevier Inc. All rights reserved.