Chk2 is a serine/threonine kinase that signals to cell cycle arrest, DNA repair, and apoptotic pathways following DNA damage. It is activated by phosphorylation in response to ionizing radiation, UV light, stalled replication forks, and other types of DNA damage. Hypoxia is a common feature of solid tumors and has been shown to affect the regulation of many genes, including several DNA repair factors. We show here that Chk2 is phosphorylated on Thr68 and thereby activated in cells in response to hypoxia, and that this phosphorylation is dependent on the damage response kinase ataxia telangiectasia mutated (ATM) but not on the related kinase ATM and Rad3-related. Moreover, phosphorylation of Chk2 under hypoxia was attenuated in cells deficient in the repair factors MLH1 or NBS1. Finally, Chk2 serves to protect cells from apoptosis under hypoxic growth conditions. These results identify hypoxia as a new stimulus for Chk2 activation in an ATM-, MLH1-, and NBS1-dependent manner, and they suggest a novel pathway by which tumor hypoxia may influence cell survival and DNA repair.