Cancer cells rely on altered metabolism to support abnormal proliferation. We performed a CRISPR/Cas9 functional genomic screen targeting metabolic enzymes and identified PDXK—an enzyme that produces pyridoxal phosphate (PLP) from vitamin B6—as an acute myeloid leukemia (AML)-selective dependency. PDXK kinase activity is required for PLP production and AML cell proliferation, and pharmacological blockade of the vitamin B6 pathway at both PDXK and PLP levels recapitulated PDXK disruption effects. PDXK disruption reduced intracellular concentrations of key metabolites needed for cell division. Furthermore, disruption of PLP-dependent enzymes ODC1 or GOT2 selectively inhibited AML cell proliferation and their downstream products partially rescued PDXK disruption induced proliferation blockage. Our work identifies the vitamin B6 pathway as a pharmacologically actionable dependency in AML. Display omitted •CRISPR/Cas9 screen identifies PDXK as an AML selective metabolic dependency•PDXK kinase activity and PLP are selectively required for leukemic cell proliferation•PLP-dependent enzymes ODC1 and GOT2 selectively support leukemic cell proliferation•The vitamin B6 pathway is a therapeutically actionable dependency in leukemia In a CRISPR/Cas9 functional screen targeting metabolic enzymes, Chen et al. identify PDXK, which produces pyridoxal phosphate (PLP) from vitamin B6, as an AML dependency. PLP-dependent enzymes ODC1 and GOT2 support AML proliferation. Blockade of the vitamin B6 metabolic pathway exhibits anti-leukemic activity.