Prenatal alcohol exposure (PAE) affects embryonic development, causing a variable fetal alcohol spectrum disorder (FASD) phenotype with neurodevelopmental disorders and birth defects. To explore the effects of PAE on gastrulation, we used an in vitro model with subchronic moderate (20†mM) and severe (70†mM) ethanol exposures during the differentiation of human embryonic stem cells into germ layer cells. We analyzed genome-wide gene expression (mRNA sequencing), DNA methylation (EPIC Illumina microarrays) and metabolome (non-targeted LC-MS) of the endodermal, mesodermal and ectodermal cells. The largest number of ethanol-induced alterations were observed in endodermal cells, whereas the most prominent changes were in ectodermal cells. Methionine metabolism and genes of the main signaling pathways involved in gastrulation and body patterning were affected by ethanol in all germ layers. Many of the altered genes, including BMP4, FGF8, SIX3 and LHX2, have previously been associated with PAE and phenotypes of FASD, like defects in heart and corpus callosum development as well as holoprosencephaly. Our findings support the early origin of alcohol-induced developmental disorders and strengthen the role of methionine cycle in the etiology of FASD.