Abstract
Metabolism regulates cell fates during early mammalian cell differentiation. This protocol describes the steps for directed differentiation of primed human pluripotent stem cells (hPSCs) into the three primary germ lineages-ectoderm, endoderm, and mesoderm-using a chemically defined nutrient-balanced media formulation. Although the transient removal and addition of specific nutrients does not occur in vivo during embryonic development, manipulation of nutrients in vitro provides an accessible method for evaluating how extracellular and intracellular metabolites help determine hPSC fate. For complete details on the use and execution of this protocol, please refer to Lu et al. (2019) and Lu et al. (2022).