Cyclin-dependent kinase (cdk) inhibitors have the potential to induce growth arrest and apoptosis in cancer cells. The genes encoding cdks involved in G1-S progression are often amplified in B-cell malignancies, including diffuse large B-cell lymphoma (DLBCL). Here, we evaluated the in vitro cytotoxic activity of the cdk2 inhibitor CVT-313 against several human DLBCL cells. Treatment of DLBCL cells with CVT-313 resulted in apoptosis. CVT-313 treatment reduced cdk2-mediated phosphorylation of the retinoblastoma gene product (Rb) on T821, but did not affect cyclin D-cdk4/6-mediated Rb phosphorylation on S807/811. Depletion of endogenous cdk2 by short interfering (si)RNA also resulted in apoptosis in human LY3, LY8 and LY18 DLBCL cells. Importantly, inhibition of cdk2 with CVT-313 or knockdown of endogenous cdk2 with siRNA resulted in down-regulation of the anti-apoptotic factor Myeloid cell leukemia-1 (Mcl-1), suggesting that decreased levels of cellular Mcl-1 contribute to apoptosis. In support of this, siRNA-mediated knockdown of Mcl-1 was sufficient to induce apoptosis in LY3 and LY18 DLBCL. Further, cdk2 inhibition led to decreased Mcl-1 mRNA levels, which was proceeded by reduced phosphorylation of serine 2 on the carboxyl terminal domain (CTD) of RNA polymerase II. Taken together, these data suggest that cdk2 activity is necessary for the survival of human DLBCL.