Abstract: Objective Evaluating important factors which have effects on cardiomyocyte differentiation to optimize differentiation efficiency. Methods Hanging drop method or 96-well plate method was used to induce aggregates. DMSO was used to induce cardiomyocyte differentiation of P19 cells. Beating cells were detected with microscopy and cardiomyocyte specific protein Troponin T was examined by immunostaining. RT-PCR was applied to evaluating the expression of cardiomyocyte related genes. Results Compared with Hanging drop method, 96-well plate method had better effect for aggregate formation and adhesion. Adding DMSO improved aggregates integrity. Seeding concentration of 1x104~2x105 cells/ml caused the most effective differentiation. Troponin T immunostaining could be observed before cells started beating. Conclusion By optimizing the method for aggregates formation, differentiation inducing chemicals and cell seeding concentration, it is possible to improve cardiomyocyte differentiation effect of P19 cells. This might provide us a way for cardiomyocyte differentiation with high efficiency.