Senescence is a potential tumor-suppressing mechanism and a commonly used model of cellular aging. One current hypothesis to explain senescence, based in part on the correlation of oxygen with senescence, postulates that it is caused by oxidative damage from reactive oxygen species (ROS). Here, we further test this theory by determining the mechanisms of hyperoxia-induced senescence. Exposure to 70% O(2) led to stress-induced, telomere-independent senescence. Although hyperoxia elevated mitochondrial ROS production, overexpression of antioxidant proteins was not sufficient to prevent hyperoxia-induced senescence. Hyperoxia activated AMPK; however, overexpression of a kinase-dead mutant of LKB1, which prevented AMPK activation, did not prevent hyperoxia-induced senescence. Knocking down p21 via shRNA, or suppression of the p16/pRb pathway by either BMI1 or HPV16-E7 overexpression, was also insufficient to prevent hyperoxia-induced senescence. However, suppressing p53 function resulted in partial rescue from senescence, suggesting that hyperoxia-induced senescence involves p53. Suppressing both the p53 and pRb pathways resulted in almost complete protection, indicating that both pathways cooperate in hyperoxia-induced senescence. Collectively, these results indicate a ROS-independent but p53/pRb-dependent senescence mechanism during hyperoxia.