Endocrine-disrupting chemicals (EDCs) are structures similar to steroids hormones which can interfere with hormone synthesis and normal physiological functions of male and female reproductive organs. EDCs tend to bind to steroid hormone receptors. Sex steroid hormones influence calcium signaling of the cardiac muscle in early embryo-development. Progesterone (P4) has been reported to affect both blood pressure and other aspects of the cardiovascular system. To confirm the effect of progesterone (P4), octyl-phenol (OP) and bisphenol A (BPA) on early differentiation of mouse embryonic stem (mES) cells into cardiomyocytes, the hanging-drop method was performed to form embryoid bodies. The mouse embryoid bodies (mEB) were suspended, attached onto 6 well plates and cultured in differentiation medium containing steroid-free FBS without LIF. P4, OP and BPA were treated at two days after attachment and media were replaced every two days. To investigate the calcium signaling, the mRNA level of calcium channel genes such as Trpv2 and contraction-related genes such as Ryr2, Cam2 and Mlck3 was analyzed. In addition, mifepristone (RU486), which is a synthetic steroid that has an affinity for progesterone receptor (PR), was used to confirm the impact of P4 through PR. To determine if RU486 is capable of attenuating the inhibition effect, RU486 was applied for one day starting on day 11. The Pgr mRNA level was significantly increased in P4, OP and BPA-treated group. However, the calcium channel genes such as Trpv2 mRNA level was significantly decreased in the P4, OP and BPA-treated group. In addition, expression of contraction-related genes such as Ryr2, Cam2 and Mlck3 were significantly decreased in the P4, OP and BPA-treated group. Interestingly, treatment of RU486 rescues altered calcium channel genes and contraction-related genes. Taken together, these results suggest that OP and BPA may impact on differentiation of mESCs into cardiomyocytes, and disrupts differentiation of cardiomyocytes.