Metabolic pathways can influence cell fate decisions, yet their regulative role during embryonic development remains poorly understood. Here, we demonstrate an instructive role of glycolytic activity in regulating signaling pathways involved in mesoderm and endoderm specification. Using a mouse embryonic stem cell (mESC)-based in vitro model for gastrulation, we found that glycolysis inhibition increases ectodermal cell fates at the expense of mesodermal and endodermal lineages. We demonstrate that this relationship is dose dependent, enabling metabolic control of germ layer proportions through exogenous glucose levels. We further show that glycolysis acts as an upstream regulator of Nodal and Wnt signaling and that its influence on cell fate specification can be decoupled from its effects on growth. Finally, we confirm the generality of our findings using a human gastrulation model. Our work underscores the dependence of signaling pathways on metabolic conditions and provides mechanistic insight into the nutritional regulation of cell fate decision-making.