Loss of RASSF1A leads to several mitotic abnormalities, including cytokinesis failure and tetraploidization. Uncontrolled proliferation of tetraploid cells is known to trigger genomic instability and tumor development and is normally prevented through activation of a p53-dependent tetraploidy checkpoint. RASSF1A is the most commonly silenced and p53 is the most frequently mutated tumor suppressor gene in human cancer. However, their mutual contribution to tumorigenesis has never been investigated in animal models. Here, we explore whether concomitant loss of RASSF1A and p53 will result in increased levels of aneuploidy, genomic instability and tumorigenesis. We have intercrossed Rassf1a-knockout mice with mice lacking the p53 gene and generated a combination of single- and compound-mutant animals. Rassf1a(-/-) p53(-/-) mice were viable and fertile and developed normally. However, these mice were remarkably tumor prone and succumbed to malignancies significantly faster than single-mutant littermates, with a median survival time of 136 days (versus 158 days in p53(-/-) mice, P=0.0207, and >600 days in Rassf1a(-/-) animals, P<0.0001). Rassf1a-null mice with one functional p53 allele displayed a more moderate, yet tumor-prone phenotype, characterized by increased tumor multiplicity as compared with single knockouts. On cell-cycle profiling and cytogenetic analysis, cells derived from Rassf1a(-/-) p53(-/-) mice exhibited several mitotic defects associated with high levels of tetraploidy/aneuploidy. Conversely, cells with a proficient p53 allele could better cope with the mitotic failures imposed by Rassf1a loss. Altogether, we provide the first experimental evidence for a pivotal role of Rassf1a as an early 'gatekeeper' gene, whose loss of function deteriorates cellular fitness by enhancing tetraploidization. Concomitant loss of p53, which causes unrestrained propagation of tetraploids into aneuploid cells, further undermines genomic stability and accelerates tumorigenesis.