Mcl-1 is an antiapoptotic Bcl-2 family member, whose degradation is supposedly required for the induction of apoptosis. However, histone deacetylase inhibitors (HDACi) induce apoptosis primarily through the Bak/Mcl-1/Noxa and Bim pathways without decreasing Mcl-1. To investigate this discrepancy, we examined the role of Mcl-1 on HDACi-mediated apoptosis. Inhibition of either class I or class II HDAC by selective HDACi caused an upregulation of Mcl-1 mRNA and protein. Downregulation of Mcl-1 by three structurally unrelated cyclin-dependent kinase inhibitors potentiated HDACi-mediated apoptosis in primary chronic lymphocytic leukemic (CLL) cells and K562 cells. Sensitivity to HDACi-induced apoptosis was increased approximately 10-fold by the cyclin-dependent kinase inhibitors. Nanomolar concentrations of HDACi, approximately 300-fold lower than that required to induce apoptosis alone, sensitized cells to TRAIL, emphasizing that the mechanism(s) whereby HDACi induce apoptosis is clearly distinct from those by which they sensitize to TRAIL. Furthermore, knockdown of Mcl-1-potentiated HDACi-mediated apoptosis in K562 cells. Thus, HDACi-mediated Mcl-1 upregulation plays an important antiapoptotic regulatory role in limiting the efficacy of HDACi-induced apoptosis, which can be overcome by combination with an agent that downregulates Mcl-1. Thus, a clinical trial in some cancers is warranted using a combination of an HDACi with agents that downregulate Mcl-1.