Pluripotent human embryonic stem (ES) cells provide a consistent developmental model for studying contaminant-induced changes in human cell fate and early developmental processes. Thus far, no studies have investigated the effects of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) exposure on the growth and differentiation of human ES cells. Here we show that the aryl hydrocarbon receptor (AhR) pathway is functional in human ES cells based on the ability of TCDD to induce the AhR/Arnt-dependent transcription of CYP1A1. No changes in colony morphology were observed in undifferentiated human ES cells following 8 days of treatment with TCDD. However, when human ES cells were allowed to replicate and spontaneously differentiate for 15 days in the presence of TCDD, colonies exhibited less morphological differentiation compared to control human ES cell cultures. This observation was confirmed on the molecular level by lower expression of markers of meso- and endodermal differentiation and higher expression of the pluripotency marker, Oct4, in TCDD-treated cultures. Additionally, TCDD inhibited the fibroblastic morphology resulting from the epithelial to mesenchymal transition (EMT) of spontaneously differentiating ES cells, preserving an undifferentiated cellular phenotype. Furthermore, lower expression of EMT markers as well as a lower incidence of the mesenchymal marker, N-cadherin, was observed around the edges of TCDD-treated cell colonies. These findings suggest that TCDD treatment inhibits human ES cell differentiation, potentially through the inhibition of an EMT-like process.