Benzo[a]pyrene (B[a]P) is one of the most common polycyclic aromatic hydrocarbons. In utero B[a]P exposure exerts multiple adverse effects on embryo development, although the underlying molecular mechanisms have still not been clearly elucidated. In the present study, we used human embryonic stem cell derived embryoid body (EB) as an in vitro model to investigate the embryotoxicity effects of B[a]P. EBs were exposed to B[a]P for 14 days, and the morphology, viability and differentiation of the cells were analyzed, in addition to the molecular changes. The results showed that B[a]P exposure repressed cell growth, impaired the morphology, and triggered apoptosis in the EBs. In addition, B[a]P reduced the gene expression levels of the ectoderm, mesoderm and endoderm biomarkers. Finally, B[a]P inhibited the epithelial-mesenchymal transition (EMT) process and the Akt/GSK-3β signaling pathway. Taken together, B[a]P-induced aberrant EB development and apoptosis were related to EMT process and the Akt/GSK-3β signaling pathway modulation.