The impressive selectivity and efficacy of BH3 mimetics for treating cancer has largely been limited to BCL-2 dependent hematological malignancies. Most solid tumors depend on other anti-apoptotic proteins, including MCL-1, for survival. The recent description of S63845 as the first specific and potent MCL-1 inhibitor represents an important therapeutic advance, since MCL-1 is not targeted by the currently available BH3 mimetics, Navitoclax or Venetoclax, and is commonly associated with chemoresistance. In this study, we confirm a high binding affinity and selectivity of S63845 to induce apoptosis in MCL-1-dependent cancer cell lines. Furthermore, S63845 synergizes with other BH3 mimetics to induce apoptosis in cell lines derived from both hematological and solid tumors. Although the anti-apoptotic BCL-2 family members in these cell lines interact with a spectrum of pro-apoptotic BH3-only proteins to regulate apoptosis, these interactions alone do not explain the relative sensitivities of these cell lines to BH3 mimetic-induced apoptosis. These findings necessitated further investigation into the requirement of BH3-only proteins in BH3 mimetic-mediated apoptosis. Concurrent inhibition of BCL-XL and MCL-1 by BH3 mimetics in colorectal HCT116 cells induced apoptosis in a BAX- but not BAK-dependent manner. Remarkably this apoptosis was independent of all known BH3-only proteins. Although BH3-only proteins were required for apoptosis induced as a result of BCL-XL inhibition, this requirement was overcome when both BCL-XL and MCL-1 were inhibited, implicating distinct mechanisms by which different anti-apoptotic BCL-2 family members may regulate apoptosis in cancer.