Establishment of primary mouse embryo fibroblasts (MEFs) as continuously growing cell lines is normally accompanied by loss of the p53 or p19^ARF tumor suppressors, which act in a common biochemical pathway. myc rapidly activates ARF andp53 gene expression in primary MEFs and triggers replicative crisis by inducing apoptosis. MEFs that survive mycoverexpression sustain p53 mutation or ARF loss during the process of establishment and become immortal. MEFs lackingARF or p53 exhibit an attenuated apoptotic response tomyc ab initio and rapidly give rise to cell lines that proliferate in chemically defined medium lacking serum. Therefore,ARF regulates a p53-dependent checkpoint that safeguards cells against hyperproliferative, oncogenic signals.