Oxygen sensitivity significantly limits the replicative lifespan of murine fibroblasts. The study compares the senescence of human and mouse embryonic fibroblasts (MEFs) under different oxygen conditions. MEFs do not senesce in physiological (3%) oxygen but undergo spontaneous immortalization in 20% oxygen. DNA damage limits MEF proliferation in 20% oxygen, as MEFs accumulate more DNA damage than human fibroblasts under these conditions. The study identifies oxygen sensitivity as a critical difference between mouse and human cells, explaining their proliferative differences in culture and possibly their different rates of cancer and aging. Replicative senescence limits cell proliferation in culture and in vivo. Human cells senesce due to telomere shortening, while MEFs senesce despite having long telomeres. MEF senescence relies on the p19ARF/p53 pathway, whereas human fibroblasts require loss of both p53 and Rb functions for immortalization. MEFs senesce after fewer population doublings than human fibroblasts, despite having longer telomeres and constitutively expressing telomerase. These differences suggest that MEFs senesce due to culture stress. The study shows that MEFs cultured in 3% oxygen do not senesce and can proliferate indefinitely, unlike those in 20% oxygen. MEFs cultured in 3% oxygen retain normal growth control and do not exhibit replicative senescence. The study also shows that MEFs cultured in 3% oxygen are sensitive to 20% oxygen for 10–15 population doublings, after which they accumulate 20% oxygen-unresponsive variants. The study identifies that DNA damage, particularly oxidative DNA damage, is a major cause of MEF replicative senescence. MEFs cultured in 3% oxygen show normal cell-cycle arrest after DNA damage and retain telomerase expression. The study also shows that MEFs cultured in 3% oxygen retain p19ARF and p53 regulation and function, unlike those in 20% oxygen. The study shows that MEFs cultured in 3% oxygen have intact Rb pathways and do not exhibit inactivation of Rb or p53. The ability to proliferate with high levels of p16 and p19ARF may entail an adaptive response or mutation of an uncharacterized pathway that allows proliferation in 20% oxygen. The study also shows that DNA repair deficiencies in mice, such as in Ku80 or DNA-PKcs, lead to increased chromosome fragmentation and fusions in 20% oxygen. The study confirms that telomere dysfunction is not a major cause of MEF replicative senescence, but oxidative DNA damage and its associated chromosomal aberrations are most likely responsibleOxygen sensitivity significantly limits the replicative lifespan of murine fibroblasts. The study compares the senescence of human and mouse embryonic fibroblasts (MEFs) under different oxygen conditions. MEFs do not senesce in physiological (3%) oxygen but undergo spontaneous immortalization in 20% oxygen. DNA damage limits MEF proliferation in 20% oxygen, as MEFs accumulate more DNA damage than human fibroblasts under these conditions. The study identifies oxygen sensitivity as a critical difference between mouse and human cells, explaining their proliferative differences in culture and possibly their different rates of cancer and aging. Replicative senescence limits cell proliferation in culture and in vivo. Human cells senesce due to telomere shortening, while MEFs senesce despite having long telomeres. MEF senescence relies on the p19ARF/p53 pathway, whereas human fibroblasts require loss of both p53 and Rb functions for immortalization. MEFs senesce after fewer population doublings than human fibroblasts, despite having longer telomeres and constitutively expressing telomerase. These differences suggest that MEFs senesce due to culture stress. The study shows that MEFs cultured in 3% oxygen do not senesce and can proliferate indefinitely, unlike those in 20% oxygen. MEFs cultured in 3% oxygen retain normal growth control and do not exhibit replicative senescence. The study also shows that MEFs cultured in 3% oxygen are sensitive to 20% oxygen for 10–15 population doublings, after which they accumulate 20% oxygen-unresponsive variants. The study identifies that DNA damage, particularly oxidative DNA damage, is a major cause of MEF replicative senescence. MEFs cultured in 3% oxygen show normal cell-cycle arrest after DNA damage and retain telomerase expression. The study also shows that MEFs cultured in 3% oxygen retain p19ARF and p53 regulation and function, unlike those in 20% oxygen. The study shows that MEFs cultured in 3% oxygen have intact Rb pathways and do not exhibit inactivation of Rb or p53. The ability to proliferate with high levels of p16 and p19ARF may entail an adaptive response or mutation of an uncharacterized pathway that allows proliferation in 20% oxygen. The study also shows that DNA repair deficiencies in mice, such as in Ku80 or DNA-PKcs, lead to increased chromosome fragmentation and fusions in 20% oxygen. The study confirms that telomere dysfunction is not a major cause of MEF replicative senescence, but oxidative DNA damage and its associated chromosomal aberrations are most likely responsible