Fatty acid metabolism governs multiple intracellular signaling pathways in many cell types, but its role in hematopoietic stem cells (HSCs) is largely unknown. Herein, we establish a critical role for 12/15-lipoxygenase (12/15-LOX)–mediated unsaturated fatty acid metabolism in HSC function. HSCs from 12/15-LOX–deficient mice are severely compromised in their capacity to reconstitute the hematopoietic compartment in competitive and serial reconstitution assays. Furthermore, we demonstrate that 12/15-LOX is required for the maintenance of long-term HSC quiescence and number. The defect in HSCs is cell-autonomous and associated with a selective reduction in 12/15-LOX–mediated generation of bioactive lipid mediators and reactive oxygen species and with a decrease in canonical Wnt signaling as measured by nuclear β-catenin staining. These results have implications for development, aging, and transformation of the hematopoietic compartment.