The emergence of cancer from diverse normal tissues has long been rationalized to represent a common set of fundamental processes. However, these processes are not fully defined. Here, we show that forced expression of glucose-6-phosphate dehydrogenase (G6PD) affords immortalized mouse and human cells anchorage-independent growth in vitro and tumorigenicity in animals. Mechanistically, G6PD augments the NADPH pool by stimulating NAD kinase-mediated NADP biosynthesis in addition to converting NADP to NADPH, bolstering antioxidant defense. G6PD also increases nucleotide precursor levels through the production of ribose and NADPH, promoting cell proliferation. Supplementation of antioxidants or nucleosides suffices to convert immortalized mouse and human cells into a tumorigenic state, and supplementation of both is required when their overlapping metabolic consequences are minimized. These results suggest that normal cells have a limited capacity for redox balance and nucleotide synthesis, and overcoming this limit might represent a key aspect of oncogenic transformation.