Human trophoblast cultures provide powerful tools to model key processes of placental development. In vitro trophoblast studies to date have relied on commercial media which contains non-physiological levels of nutrients, and the impact of these conditions on trophoblast metabolism and function is unknown. Here we show that the physiological medium (Plasmaxä) with nutrient and metabolite concentrations recapitulating human plasma improves human trophoblast stem cell (hTSC) proliferation and differentiation compared to standard medium (DMEM-F12). hTSCs cultured in Plasmax-based medium also show altered glycolytic and mitochondrial metabolism, as well as reduced S-adenosylmethionine/S-adenosyl-homosysteine ratio compared to DMEM-F12-based medium. These findings demonstrate the importance of the nutritional environment for phenotyping cultured human trophoblasts.