Xenoestrogens can have adverse effects on the reproductive and immune systems; for example, 4-tert-octylphenol (OP) and 4-nonylphenol (NP) can have estrogenic effects in target cells. In this study, we investigated the effects of xenoestrogens on the expression of undifferentiation and differentiation markers in mouse embryonic stem (ES) cells, which are important mediators of the differentiation of ES cells into cardiomyocytes. The ES cells were treated with 17β-estradiol or OP and NP in a time-dependent manner (for 1, 2, or 3 days), and embryoid bodies (EB) cells were given the same treatment for 5, 8, 12, or 16 days. The mRNA expressions of undifferentiation markers (Oct-4, Sox2, Zfp206, and Rex-1) and cardiomyocyte differentiation markers (α-MHC, β-MHC, ANF, and MLC-2V) were determined by semi- and quantitative real-time PCR. Treatment with E2 induced an increase (1.3- to 4.6-fold) in Oct-4 expression at the transcriptional level in a dose- and time-dependent manner. However, no difference was observed in the expression of Sox2, Zfp206, or Rex-1 genes in ES cells, suggesting that E2 might be an Oct-4 enhancer in ES cells. However, induction of Oct-4 expression by E2 might result from changes in the Oct-4 promoter methylation pattern rather than from other regulatory mechanisms. We also found that cardiomyocyte differentiation markers were differentially expressed in response to xenoestrogens in EB cells. Taken together, these results suggest that xenoestrogens might play a role as a positive regulator of the undifferentiation process in mouse ES and EB cells and might be involved in the maintenance and differentiation of mouse ES cells.