Tetraploidization is believed to promote genome instability and tumorigenesis. Whether tetraploids per se are intrinsically unstable and transforming remain incompletely understood. In this report, tetraploidization was induced with cell fusion using mouse fibroblasts. Due to the unequal segregation of chromosomes during multipolar mitosis, the majority of cells were eliminated by p53-dependent mechanisms after tetraploidization. The rare tetraploid fibroblasts that were able to undergo bipolar mitosis remained chromosomally stable and nontransformed over many generations. Suppression of p53 functions during tetraploidization, either by RNA interference or by using p53-deficient mouse fibroblasts, produced cells that were chromosomally unstable. They were fast growing and displayed anchorage-independent growth in soft agar. In contrast, impairment of p53 functions after tetraploids were established was ineffective in triggering chromosomal instability and transformation. Collectively, these results are consistent with a model that during early stages of tetraploidization, the lack of p53 promotes the survival of chromosomally unstable sub-tetraploids, leading to transformation. Once tetraploids are established, however, p53 is not essential for maintaining chromosome stability.