Lipoprotein lipase (LPL) mediates hydrolysis of triglycerides (TGs) to supply free fatty acids (FFAs) to tissues. Here, we show that LPL activity is also required for hematopoietic stem progenitor cell (HSPC) maintenance. Knockout of Lpl or its obligatory cofactor Apoc2 results in significantly reduced HSPC expansion during definitive hematopoiesis in zebrafish. A human APOC2 mimetic peptide or the human very low-density lipoprotein, which carries APOC2, rescues the phenotype in apoc2 but not in lpl mutant zebrafish. Creating parabiotic apoc2 and lpl mutant zebrafish rescues the hematopoietic defect in both. Docosahexaenoic acid (DHA) is identified as an important factor in HSPC expansion. FFA-DHA, but not TG-DHA, rescues the HSPC defects in apoc2 and lpl mutant zebrafish. Reduced blood cell counts are also observed in Apoc2 mutant mice at the time of weaning. These results indicate that LPL-mediated release of the essential fatty acid DHA regulates HSPC expansion and definitive hematopoiesis.