L-proline stimulates transition of mouse embryonic stem cells (ESCs) to early primitive ectoderm-like cells (EPLCs) by activating signalling pathways. We show changes in cellular metabolism are also required for the transition: the L-proline-mediated transition depends on the pentose phosphate pathway (PPP) which stimulates glycolysis, resulting in increasing anaerobic metabolism. The role of the PPP in the ESC-to-EPLC transition was assessed by culturing ESCs with L-proline to produce EPLCs with or without 6-aminonicotidamide (6AN) and dichloroacetate (DCA). 6AN is a competitive substrate of glucose-6-phosphate dehydrogenase, the rate-limiting enzyme of the PPP. DCA inhibits pyruvate dehydrogenase kinase, alleviating inhibition of pyruvate dehydrogenase to allow pyruvate to enter mitochondrial respiration, thus decreasing the rate of anaerobic metabolism via glucose. When 6AN or DCA was added to ESCs along with L-proline, it prevented the decrease in doubling time and reduced the percentage of differentiated colonies expected to occur with L-proline alone. 6AN or DCA also prevented L-proline-mediated upregulation of EPLC markers Fgf5 and Dmnt3b. Addition of L-proline to ESCs increased anaerobic metabolism rate but addition of 6AN or DCA prevented this increase. Therefore, an increase in anaerobic metabolism involving the PPP along with L-proline-mediated activation of signalling pathways is required for L-proline-mediated ESC-to-EPLC transition.