Replicative senescence is induced by critical telomere shortening and limits the proliferation of primary cells to a finite number of divisions. To characterize the activity status of the replicative senescence program in the context of cell cycle activity, we analyzed the senescence phenotypes and signaling pathways in quiescent and growth-stimulated primary human fibroblasts in vitro and liver cells in vivo. This study shows that replicative senescence signaling operates at a low level in cells with shortened telomeres but becomes fully activated when cells are stimulated to enter the cell cycle. This study also shows that the dysfunctional telomeres and nontelomeric DNA lesions in senescent cells do not elicit a DNA damage signal unless the cells are induced to enter the cell cycle by mitogen stimulation. The amplification of senescence signaling and DNA damage responses by mitogen stimulation in cells with shortened telomeres is mediated in part through the MEK/mitogen-activated protein kinase pathway. These findings have implications for the further understanding of replicative senescence and analysis of its role in vivo.