Hypoxia inducible factor (HIF)-1 is a master transcriptional regulator mediating the cellular adaptation to hypoxia. In addition, HIF-1 is also vital for the development of hematopoietic stem cells (HSCs). In a previous study we found that thrombopoietin (TPO), an important and non-redundant cytokine for HSC maintenance and expansion, induces HIF-1α expression in HSCs by enhancing the stability of HIF-1α under normoxic conditions. However, the molecular mechanisms of these effects are not yet fully understood. In this study, we explored the mechanisms and found that TPO-induced mitochondrial reactive oxygen species (ROS) played a crucial role in stabilization of HIF-1. Both ROS scavengers and inhibitors of mitochondrial electron transport completely blocked HIF-1α induction by TPO in UT-7/TPO cells and in primary immature mouse bone marrow cells. We also found that TPO-induced HIF-1α induction was tightly coupled with glucose metabolism. Inhibition of glucose transporter or glycolytic enzyme blocked HIF-1α elevation of TPO. These results indicate that TPO induces HIF-1α expression in a manner very similar to that of hypoxia.