Pharmacologic or genetic inhibition of MTHFD2 markedly suppresses leukemia progression both in vitro and in vivo by constraining one-carbon metabolism, leading to deoxynucleotide and thymidylate depletion, replication stress, S-phase arrest, and subsequent apoptotic cell death, thereby delineating a therapeutically exploitable metabolic vulnerability. This evidence concerns the gene MTHFD2 and leukemia.