Senescence of endothelial cells increases with systemic aging and is thought to contribute to the development of atherosclerosis. Cell therapy with highly proliferative endothelial progenitor cells (EPCs) is an emerging therapeutic option to promote endothelial regeneration, but little is known about their senescence and their vulnerability to inflammatory stressors. We therefore studied the senescence of proliferative human EPCs and investigated the effects of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) on their senescence. Human EPCs had a significantly lower rate of senescence at baseline, compared with that of mature endothelial cells. However, EPCs up-regulated the expression of the senescence-associated cell cycle arrest protein p16(INK4a) and markedly increased measured senescence levels when exposed to chronic TNF-alpha treatment. Analysis of telomere length showed that the increases in senescence were not related to changes in telomere length. Inhibition of the p38 mitogen-activated protein kinase pathway blocked the induction of p16(INK4a) and cellular senescence. In conclusion, highly proliferative EPCs have a low rate of intrinsic senescence but are vulnerable to premature senescence induction by chronic proinflammatory stimulation. These findings will lead to a better understanding of physiological endothelial regeneration as well as to targeted therapies with the aim of promoting endothelial regeneration through endothelial progenitor cells.