During embryonic development, cells exit pluripotency to give rise to the three germ layers. Metabolic pathways influence cell fate decisions by modulating the epigenetic, transcriptional, and signalling states of cells. However, the interplay between metabolism and the major signalling pathways that drive the emergence of ectoderm, mesoderm, and endoderm remains poorly understood. Here, we demonstrate an instructive role of glycolytic activity in activating signalling pathways involved in mesoderm and endoderm induction. Using an in vitro model system for mouse gastrulation, we observed that inhibiting glycolysis prevents the upregulation of primitive streak markers, resulting in a significant increase in 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. Mechanistically, we found that glycolysis inhibition leads to the downregulation of Wnt, Nodal, and Fgf signalling. Notably, this metabolic phenotype was rescued by Nodal or Wnt signalling agonists in the absence of glycolytic activity, suggesting that glycolytic activity acts upstream of both signalling pathways. Our work underscores the dependence of specific signalling pathways on metabolic conditions and provides mechanistic insight into the nutritional regulation of cell fate decision making.

Metabolic control of germ layer proportions through regulation of Nodal and Wnt signalling