Participation of metabolism in stem cell differentiation has been largely disregarded until recently. Here, functional proteomics and metabolomics were performed to unveil the mitochondrial and nuclear metabolism during dimethyl sulfoxide (DMSO)-induced differentiation of P19 cells. DMSO-treated cells were shown to exhibit increased glycolytic enzymes activities and fuel pyruvate into oxidative phosphorylation. Subsequently, enzymes of electron transport chain also had elevated activities upon differentiation. These changes in mitochondrial metabolism were concomitant with increased mitochondrial biogenesis as PGC-1α expression was higher in the differentiated cells. To study nuclear metabolism, particular focus was placed on delineating a potential role of nuclear lactate dehydrogenase (LDH). Nuclear LDH was found to exhibit higher expression in pluripotent cells. NAD+ generated from LDH reaction was discovered to promote histone deacetylation via sirtuin-1 (SIRT1). Drastic alterations in mitochondrial and nuclear metabolism during differentiation point to a pivotal role of metabolism in deciding the final destination of stem cells.