Establishment of cell lines from primary mouse embryo fibroblasts depends on loss of either the Arf tumor suppressor or its downstream target, the p53 transcription factor. Mouse p19^Arf is encoded by the Ink4a-Arf locus, which also specifies a second tumor suppressor protein, the cyclin D-dependent kinase inhibitor p16^Ink4a. We surveyed bone marrow-derived cells from wild-type, Ink4a-Arf-null, or Arf-null mice for their ability to bypass senescence during continuous passage in culture. Unlike preB cells from wild-type mice, those from mice lacking Arf alone could be propagated indefinitely when placed onto stromal feeder layers engineered to produce IL-7. The preB cell lines remained diploid and IL-7-dependent and continued to express elevated levels of p16^Ink4a. By contrast, Arf-null bone marrow-derived macrophages that depend on colony-stimulating factor-1 for proliferation and survival in culture initially grew at a slow rate but gave rise to rapidly and continuously growing, but still growth factor-dependent, variants that ceased to express p16^Ink4a. Wild-type bone marrow-derived macrophages initially expressed both p16^Ink4a and p19^Arf but exhibited an extended life span when p16^Ink4a expression was extinguished. In all cases, gene silencing was accompanied by methylation of the Ink4a promoter. Therefore, whereas Arf loss alone appears to be the major determinant of establishment of murine fibroblast and preB cell lines in culture, p16^Ink4a provides an effective barrier to immortalization of bone marrow-derived macrophages.