Key Points• PGE 2 signaling positively regulates hematopoietic stem cells both directly and via activation of a nonhematopoietic cell population.• EP4 is a major receptor for the PGE 2 -mediated regulation of hematopoietic stem and progenitor cells.Prostaglandin E 2 (PGE 2 ) regulates hematopoietic stem/progenitor cell (HSPC) activity. However, the receptor(s) responsible for PGE 2 signaling remains unclear. Here, we identified EP4 as a receptor activated by PGE 2 to regulate HSPCs. Knockdown of Ep4 in HSPCs reduced long-term reconstitution capacity, whereas an EP4-selective agonist induced phosphorylation of GSK3b and b-catenin and enhanced long-term reconstitution capacity. Next, we analyzed the niche-mediated effect of PGE 2 in HSPC regulation. Bone marrow mesenchymal progenitor cells (MPCs) expressed EP receptors, and stimulation of MPCs with PGE 2 significantly increased their ability to support HSPC colony formation. Among the EP receptor agonists, only an EP4 agonist facilitated the formation of HSPC colonies after the coculture with MPCs. PGE 2 up-regulated the expression of cytokine-, cell adhesion-, extracellular matrix-, and protease-related genes in MPCs. We also examined the function of PGE 2 /EP4 signaling in the recovery of the HSPCs after myelosuppression. The administration of PGE 2 or an EP4 agonist facilitated the recovery of HSPCs from 5-fluorouracil (5-FU)-induced myelosuppression, indicating a role for PGE 2 /EP4 signaling in this process. Altogether, these data suggest that EP4 is a key receptor for PGE 2 -mediated direct and indirect regulation of HSPCs. (Blood.