Glucose metabolism has a crucial role for providing substrates required to generate ATP and regulate the epigenetic landscape. We reported that F9 embryonal carcinoma stem-like cells require cytosolic reactive oxygen species to differentiate into extraembryonic endoderm; however, mitochondrial sources were not examined. To extend these studies, we examined the metabolic profile of early and late-passage F9 cells, and show that their ability to differentiate is similar, even though each population has dramatically different metabolic profiles. Differentiated early-passage cells relied on glycolysis, while differentiated late-passage cells transitioned towards oxidative phosphorylation (OXPHOS). Unexpectedly, electron transport chain protein stoichiometry was disrupted in differentiated late-passage cells, whereas genes encoding mitofusion 1 and 2, which promote mitochondrial fusion and favor OXPHOS, were upregulated in differentiated early-passage cells. Despite this, early-passage cells cultured under conditions to promote glycolysis showed enhanced differentiation, whereas promoting OXPHOS in late-passage cells showed a similar trend. Further analysis revealed that the distinct metabolic profiles seen between the two populations is largely associated with changes in genomic integrity, linking metabolism to passage number. Together, these results indicate that passaging has no effect on the potential for F9 cells to differentiate into extraembryonic endoderm; however, it does impact their metabolic profile. Thus, it is imperative to determine the molecular and metabolic status of a stem cell population before considering its utility as a therapeutic tool for regenerative medicine.