DNA damage checkpoints are critical for preventing tumorigenesis and regulating the response of cells to genotoxic agents. It is believed that the coordinated actions of a number of effectors underlie proper checkpoint function. The kinase Chk2, p21, and 14-3-3σ have each been shown to be independent effectors of the G2 DNA damage checkpoint. However, the relative roles of these proteins remain unclear. To help elucidate this question, we have perturbed each of these 3 genes in combination in human cells. We show that Chk2 depletion causes markedly increased sensitivity to DNA damage in p21-/-, 14-3-3σ-/- cells but not in cells lacking only one or none of these genes. This greater sensitivity was due to an increase in apoptosis following DNA damage and not due to exacerbation of G2 checkpoint defects. Pharmacologic inhibition of Chk2 in p21-/-, 14-3-3σ-/- cells also resulted in greater sensitivity to DNA damage. Our data indicates that p21 and 14-3-3σ synergize as molecular determinants of sensitivity to DNA damage following Chk2 inhibition, and Chk2 modulates the biological rheostat that determines whether a cancer cell undergoes arrest versus death after treatment with a chemotherapeutic agent. These findings have implications for the targeting of Chk2 in human cancers.