Prostaglandin E2 (PGE2) regulates hematopoietic stem/progenitor cell (HSPC) activity. However, the receptor(s) responsible for PGE2 signaling remains unclear. Here we identified EP4 as a receptor activated by PGE2 to regulate HSPCs. Knockdown of Ep4 in HSPCs reduced long-term reconstitution (LTR) capacity, while an EP4-selective agonist induced phosphorylation of GSK3β and β-catenin and enhanced LTR capacity. We next analyzed the niche-mediated effect of PGE2 in the HSPC regulation. Bone marrow mesenchymal progenitor cells (MPCs) expressed EP receptors, and stimulation of MPCs with PGE2 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. PGE2 upregulated the expression of cytokine-, cell adhesion-, extracellular matrix-, and protease-related genes in MPCs. We also examined the function of PGE2/EP4 signaling in the recovery of the HSPCs after myelosuppression. The administration of PGE2 or an EP4 agonist facilitated the recovery of HSPCs from 5-fluorouracil-induced myelosuppression, indicating a role for PGE2/EP4 signaling in this process. Altogether, these data suggest that EP4 is a key receptor for PGE2-mediated direct and indirect regulation of HSPCs.