The retinoblastoma (Rb) tumor suppressor gene is frequently inactivated in cancer, resulting in deregulated activation of E2F transcription factors, which promote S-phase entry, p53-dependent and p53-independent apoptosis. Transformed cells evade p53-dependent apoptosis initiated by Rb inactivation by TP53 mutation. However, the mechanisms by which cancer cells circumvent p53-independent apoptosis in this context are poorly understood. Because Rb inactivation primes cells for apoptosis by p53-independent induction of procaspases, we postulated that αB-crystallin, an inhibitor of procaspase-3 activation, would suppress caspase activation in cells with combined Rb and p53 inactivation. Notably, αB-crystallin is commonly expressed in ER/PR/HER2 "triple-negative" breast carcinomas characterized by frequent Rb loss and TP53 mutation. We report that αB-crystallin (-/-) knock out (KO) MEFs immortalized by dominant negative (DN) p53 are resistant to transformation by the adenovirus E1A oncoprotein, which inactivates Rb, while wild-type (WT) MEFs are readily transformed by DN p53 and E1A. αB-crystallin (-/-) KO MEFs stably expressing DN p53 and E1A were more sensitive to chemotherapy-induced caspase-3 activation and apoptosis than the corresponding WT MEFs, despite comparable induction of procaspases by E1A. Similarly, silencing Rb in WT and αB-crystallin (-/-) KO MEFs immortalized by DN p53 increased procaspase levels and sensitized αB-crystallin (-/-) KO MEFs to chemotherapy. Furthermore, silencing αB-crystallin in triple-negative breast cancer cells, which lack Rb and express mutant p53, enhanced chemotherapy sensitivity compared to non-silencing controls. Our results indicate that αB-crystallin inhibits caspase activation in cells primed for apoptosis by Rb inactivation and plays a novel oncogenic role in the context of combined Rb and p53 inactivation.