The intrinsic mitochondrial apoptotic pathway acts through two core pro-apoptotic proteins Bax and Bak. While Bax and Bak seem to play redundant roles in apoptosis, accumulating evidence also suggests that they might not be interchangeable under certain conditions, at least in some human cell lines. Here we report the generation of Bak knockout as well as BaxBak double knockout HCT116 human colon carcinoma cells. We show that Bak is dispensable for apoptosis induced by a variety of stimuli including ABT-737 but not for 5-FU-induced apoptosis. In addition, Bax deficiency only provides partial protection against camptothecin and cisplatin-induced apoptosis and no protection against killing by Puma or ABT-737 plus Noxa overexpression. Moreover, Bak is activated normally in response to many chemotherapeutic drugs in the presence of Bax but remains kept in check by Mcl-1 in the absence of Bax. Our data suggest that Bax and Bak are functionally redundant but they are counteracted by distinct anti-apopotic Bcl-2 family proteins in different species.