An unbiased screen for genes that can immortalize mouse embryonic fibroblasts identified the glycolytic enzyme phosphoglycerate mutase (PGM). A 2-fold increase in PGM activity enhances glycolytic flux, allows indefinite proliferation, and renders cells resistant to ras-induced arrest. Glucosephosphate isomerase, another glycolytic enzyme, displays similar activity and, conversely, depletion of PGM or glucosephosphate isomerase with short interfering RNA triggers premature senescence. Immortalized mouse embryonic fibroblasts and mouse embryonic stem cells display higher glycolytic flux and more resistance to oxidative damage than senescent cells. Because wild-type p53 down-regulates PGM, mutation of p53 can facilitate immortalization via effects on PGM levels and glycolysis.