Human embryonic stem cells are derived from the inner cell mass of preimplantation embryo at the blastocyst stage and their differentiation occurs through an intermediate step involving the formation of embryoid bodies (EBs), which are aggregates of embryonic stem cells. The EBs seem to be a powerful tool for investigating the development of embryos, as they can mimic the initial stages of embryonic development. In this study, we aimed to investigate the effect of estrogen compounds on the proliferation and differentiation of short-term and long-term cultured EBs in vitro. For this study, 10-day-old (short-term cultured) and 30-day-old (long-term cultured) EBs were subjected to estradiol (E2), estriol (E3), selective estrogen receptor modulator (raloxifene [RLX]), bisphenol A, and 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole for 7 days. To confirm the effects of estrogen treatment, ICI-182780 was added to the respective EBs for additional 7 days following estrogen treatment. Quantitative reverse transcription-polymerase chain reaction was performed to analyze the relative expression of differentiation marker genes representing the 3 germ layers. The expression of 7 marker genes, which included α-fetoprotein, hepatocyte nuclear factor (HNF)-3β, HNF-4α (endoderm), brachyury, cardiac actin ([cACT]; mesoderm), nestin (ectoderm), and Oct-4 (undifferentiated), was measured. Significantly, lower expression of HNF-4α in both short-term and long-term cultured EBs was observed after treatment of estrogen compounds compared to control. The expression of HNF-3β in short-term cultured EBs has been positively affected by E2, E3, and RLX. Regarding cACT, higher expression was observed after treatment of E2 (10(-7) mol/L) and E3 (10(-9) mol/L) in short-term cultured EBs, but opposite effects were demonstrated in long-term cultured EBs. The lower expressions of HNF-4α by E2 and RLX were negated by ICI-182780 treatment, although these findings were not statistically significant in E3-treated group. These findings suggest that estrogen compounds have effects on endodermal and mesodermal differentiation of human EBs.