Acute myeloid leukemia (AML) cells are highly dependent on glycolytic pathways to generate metabolic energy and support cell growth, hinting at specific, targetable vulnerabilities as potential novel targets for drug development. Elevated levels of NADPH, a central metabolic factor involved in redox reactions, are common in myeloid leukemia cells, but the significance or biochemical basis underlying this increase is unknown. Using a small molecule analog that efficiently inhibits NADPH-producing enzymes, we found that AML cells require NADPH homeostasis for cell growth. We also found that inhibiting NADPH production through knockdown of 6-phosphogluconate dehydrogenase (6PGD) within the pentose phosphate pathway was sufficient to reduce cell growth and lactate production, a measure of metabolic reprogramming. Further, inhibition of 6PGD activity reduced NADH levels and enzymatic activity of the oxidized NADH-dependent sirtuin-1. Targeting 6PGD and NADPH production was sufficient to block growth of AML cell lines resistant to the chemotherapeutics daunorubicin and cytarabine. Importantly, stromal cell-mediated resistance to targeted inhibition of oncogenic FLT3 kinase activity by quizartinib was circumvented by 6PGD knockdown. Overall, these data suggest that the dependency of AML cells on NADPH to permit increased glycolytic flux creates a potential vulnerability of possible therapeutic benefit, since much of the enhanced production of NADPH is dependent on the activity of a single enzyme, 6PGD.