Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor, which mediates the effects of a variety of environmental stimuli in multiple tissues. Recent advances in AHR biology have underlined its importance in cells with high developmental potency, including pluripotent stem cells. Nonetheless, there is little data on AHR expression and its role during the initial stages of stem cell differentiation. The purpose of this study was to investigate the temporal pattern of AHR expression during directed differentiation of human embryonic stem cells (hESC) into neural progenitor, early mesoderm and definitive endoderm cells. Additionally, we investigated the effect of the AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the gene expression profile in hESCs and differentiated cells by RNA-seq, accompanied by identification of AHR binding sites by ChIP-seq and epigenetic landscape analysis by ATAC-seq. We showed that AHR is differentially regulated in distinct lineages. We provided evidence that TCDD alters gene expression patterns in hESCs and during early differentiation. Additionally, we identified novel potential AHR target genes, which expand our understanding on the role of this protein in different cell types.