[Show abstract][Hide abstract] ABSTRACT: Terminally differentiated cell types are needed to live and function in a postmitotic state for a lifetime. Cellular senescence is another type of permanent arrest that blocks the proliferation of cells in response to genotoxic stress. Here we show that the retinoblastoma protein (pRB) uses a mechanism to block DNA replication in senescence that is distinct from its role in permanent cell cycle exit associated with terminal differentiation. Our work demonstrates that a subtle mutation in pRB that cripples its ability to interact with chromatin regulators impairs heterochromatinization and repression of E2F-responsive promoters during senescence. In contrast, terminally differentiated nerve and muscle cells bearing the same mutation fully exit the cell cycle and block E2F-responsive gene expression by a different mechanism. Remarkably, this reveals that pRB recruits chromatin regulators primarily to engage a stress-responsive G(1) arrest program.