Reversine is a small synthetic molecule that inhibits multiple mitotic kinases, including MPS1 as well as Aurora kinase A and B (AURKA and AURKB). Here, we investigated the effects of reversine on p53-deficient vs p53-proficient cancer cells. We found that low doses (~0.5 µM) of reversine, which selectively inhibit MPS1 and hence impair the spindle assembly checkpoint, kill human TP53 (-/-) colon carcinoma cells less efficiently than their wild-type counterparts. In sharp contrast, high doses (~5 µM) of reversine induced hyperploidization and apoptosis to a much larger extent in TP53 (-/-) than in TP53 (+/+) cells. Such a selective cytotoxicity could not be reproduced by the knockdown of MPS1, AURKA and AURKB, neither alone nor in combination, suggesting that it involves multiple (rather than a few) molecular targets of reversine. Videomicroscopy-based cell fate profiling revealed that, in response to high-dose reversine, TP53 (-/-) (but not TP53 (+/+) ) cells undergo several consecutive rounds of abortive mitosis, resulting in the generation of hyperpolyploid cells that are prone to succumb to apoptosis upon the activation of mitotic catastrophe. In line with this notion, the depletion of anti-apoptotic proteins of the BCL-2 family sensitized TP53 (-/-) cells to the toxic effects of high-dose reversine. Moreover, the knockdown of BAX or APAF-1, as well as the chemical inhibition of caspases, limited the death of TP53 (-/-) cells in response to high-dose reversine. Altogether, these results suggest that p53-deficient cells are particularly sensitive to the simultaneous inhibition of multiple kinases, including MPS1, as it occurs in response to high-dose reversine.