Endocrine-disrupting chemicals (EDCs) have structures similar to steroid hormones and can interfere with hormone synthesis and normal physiological functions of reproductive organs. For example, sex steroid hormones influence calcium signaling of the cardiac muscle in early embryo development. To confirm the effect of progesterone (P4), octyl-phenol (OP), and bisphenol A (BPA) on early differentiation of mouse embryonic stem cells (mESCs) into cardiomyocytes, mESCs were treated with P4, OP, and BPA two days after attachment and media were replaced every two days. In addition, cells were treated with mifepristone (RU486), a synthetic steroid that has an affinity for progesterone receptor (Pgr), for one day starting on day 11. Beating ratio was decreased with P4, OP, and BPA treatment. The Pgr mRNA level was significantly increased in the P4-, OP- and BPA-treated groups. However, the mRNA level of the calcium channel gene (Trpv2), contraction-related genes (Ryr2, Cam2, and Mylk3) and cardiac development and morphogenesis genes (Rbp4, Ly6e, and Gata4) were significantly decreased in the P4-, OP-, and BPA-treated groups. Interestingly, treatment with RU486 rescued the altered calcium channel gene, contraction-related genes, and cardiac development and morphogenesis genes. P4, OP, and BPA treatments reduced the intracellular calcium level. Taken together, these results indicate that EDCs (OP and BPA) has a structure similar to that of endogenous steroid hormones such as progesterone and estrogen, and OP and BPA act like progesterone to inhibit and disrupt cardiomyocyte differentiation of mESCs.