Abstract The Ink4-Arf locus encodes three tumor suppressor proteins, two of which (p16Ink4a and p15Ink4b) inhibit the activities of cyclin D-dependent kinases (Cdk4 and Cdk6) and a third (p19Arf) that inhibits the Mdm2 E3 ligase to activate p53. Arf induction in response to aberrantly increased and persistent signaling thresholds mediated by activated oncogenes triggers a p53-dependent transcriptional program that can lead to cell cycle arrest or apoptosis, thereby eliminating incipient tumor cells. Arf inactivation compromises these p53-dependent protective effects and synergizes with oncogene activation to drive tumor progression. The Ink4-Arf locus is epigenetically silenced in self-renewing stem cells by Polycomb Group (PcG) complexes but is remodeled as stem cells differentiate to yield progeny that have lost their unlimited self-renewal potential. Thus, although it is generally not engaged in most normal tissues, the Ink4-Arf locus is “poised” to respond to oncogenic stress signals in non-stem cells and can thereby act as a differentiation stage-specific tumor suppressor. Deletion of Ink4-Arf is a frequent event in many forms of cancer, helping to reconfer the ability of tumor cells to self-renew indefinitely. The fact that INK4A-ARF deletions and p53 mutations occur in a mutually exclusive manner in many human tumors provides the strongest argument that disruption of the ARF-HDM2-p53 “pathway” is a hallmark of cancer in humans as well as in mice. We have produced several strains of mice in which functional Arf coding sequences in the mouse genome have been replaced by reporter genes. These include cassettes encoding green fluorescent protein (GFP) or Cre recombinase that are driven by the cellular Arf promoter. These experiments revealed that, apart from monitoring latent oncogenic signals in living animals, the Arf promoter is transiently engaged in several normal cell types. For example, Arf is expressed in the male germ cell lineage in mitotically amplifying spermatogonia but not in the primary spermatocytes that derive from them. Notably, in this setting, Arf expression is compatible with cell division and does not trigger apoptosis or cellular senescence. However, inactivation of Arf alters the homeostatic relationship between spermatogonia and spermatocytes and leads to a progressive depletion of male germ stem cells and to precocious infertility as animals age. Surprisingly, although the Ink4a gene is co-expressed with Arf in spermatogonia, its inactivation results in increased sperm production, thereby offsetting the effects of Arf loss. In short, despite their intimate linkage and ability to be epigenetically silenced en bloc, Ink4a and Arf inactivation lead to opposing phenotypes in this setting. We suspect that the ability of transient Arf and Ink4a expression to “gate” stem cell self-renewal may reflect a primordial function of this locus distinct from its role in tumor suppression. This may potentially explain why this gene cluster is evolutionarily conserved in mammals, despite its strong propensity to undergo deletion in cancer cells. Citation Format: Charles J. Sherr, Adam Gromley, Michelle L. Churchman, Emmanuel J. Volanakis. Regulation of cellular self-renewal by the ARF [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY29-03