Summary. Background: Platelet production is an intricate process that is poorly understood. Recently, we demonstrated that the natural peroxisome proliferator-activated receptor gamma (PPARγ) ligand, 15-deoxy-Δ12,14 prostaglandin J2 (15d-PGJ2), augments platelet numbers by increasing platelet release from megakaryocytes through the induction of reactive oxygen species (ROS). 15d-PGJ2 can exert effects independent of PPARγ, such as increasing oxidative stress. Heme oxygenase-1 (HO-1) is a potent antioxidant and may influence platelet production. Objectives: To further investigate the influence of 15d-PGJ2 on megakaryocytes and to understand whether HO-1 plays a role in platelet production. Methods: Meg-01 cells (a primary megakaryoblastic cell line) and primary human megakaryocytes derived from cord blood were used to examine the effects of 15d-PGJ2 on HO-1 expression in megakaryocytes and their daughter platelets. The role of HO-1 activity in thrombopoiesis was studied using established in vitro models of platelet production. Results and conclusions: 15d-PGJ2 potently induced HO-1 protein expression in Meg-01 cells and primary human megakaryocytes. The platelets produced from these megakaryocytes also expressed elevated levels of HO-1. 15d-PGJ2-induced HO-1 was independent of PPARγ, but could be replicated using other electrophilic prostaglandins, suggesting that the electrophilic properties of 15d-PGJ2 were important for HO-1 induction. Interestingly, inhibiting HO-1 activity enhanced ROS generation and augmented 15d-PGJ2-induced platelet production, which could be attenuated by antioxidants. These new data reveal that HO-1 negatively regulates thrombopoiesis by inhibiting ROS.