Acquired resistance to targeted drugs is emerging as an obstacle to successful cancer treatment. Recently, a BCL2-selective BH3 mimetic termed ABT-199 showed promising therapeutic results in BCL2-dependent tumors. Based on its high affinity for BCL2, we studied potential mechanisms conferring resistance upon ABT-199 therapy, aiming to anticipate its occurrence in the clinic. Two models of resistant lymphomas were established by continuous ABT-199 exposure. In resistant Bcl2-expressing mouse lymphoma cells, 2 missense mutations within the Bcl2 BH3 domain were identified. Both F101C and F101L mutations impeded ABT-199 binding to the BH3 domain, therefore suppressing mitochondrial apoptosis. In resistant human lymphoma cells, a missense mutation in the C-terminal transmembrane domain of proapoptotic BAX (G179E) was found, which abrogated BAX anchoring to mitochondria and blocked ABT-199–induced apoptosis both in vitro and in vivo. Importantly, G179E BAX mutation also induced partial cross-resistance to other antineoplastic drugs. Our study reveals the acquisition of mutations in BCL2 family proteins as a novel mechanism of apoptosis resistance in cancer. These results anticipate the potential development of such mutations in patients treated with ABT-199, providing a basis to preventing their occurrence and to designing drugs able to circumvent the acquired resistance.