Erythropoietin (EPO) regulates the production of red blood cells primarily by preventing apoptosis of erythroid progenitors. More recently however, EPO has emerged as a major cytoprotective cytokine in several non-hemopoietic tissues in the setting of stress or injury. The underlying mechanisms of the protective responses of EPO have not been fully defined. Here we show that EPO triggers a phosphatidylinositol 3-kinase-(PI3K) dependent survival pathway that counteracts endothelial cell death. The protection conferred by PI3K relies on the subsequent induction of Bcl-X_L, a pro-survival member of the Bcl-2 protein family. In addition, EPO counteracts the upregulation of the pro-apoptotic BH3-only protein, BIM, which is induced by serum withdrawal. EPO also activates extracellular signal-regulated kinase 1 and 2 (ERK1/2) which are involved in a Bcl-X_L-independent cytoprotective pathway. EPO caused a prolonged activation of NF-B, which was blocked by inhibition of PI3K, but not by inhibition of MAP/ERK kinase (MEK), suggesting that EPO-activated NF-B requires PI3K activity. However, the activation of the NF-B pathway was not required for the ability of EPO to counteract endothelial apoptosis. Thus, EPO promotes survival of endothelial cells through PI3K-dependent Bcl-X_L-induction and BIM regulation, as well as through a separate mechanism involving the ERK pathway.