The 14-3-3 proteins are a family of highly conserved eukaryotic regulatory molecules that play important roles in many biological processes including cell cycle control and regulation of cell death. They are able to carry out these effects through binding and modulating the activity of a host of signaling proteins. The ability of 14-3-3 to inhibit Bad and other proapoptotic proteins argues that 14-3-3 can support cell survival. To examine this issue in a global sense, a specific inhibitor of 14-3-3/ligand interactions, difopein, was used. Difopein expression led to induction of apoptosis. Studies using various components of survival and death signaling pathways were consistent with a vital role for 14-3-3/ligand interactions in signal transduction from upstream pro-survival kinases to the core apoptotic machinery. Because these kinases often become activated during oncogenesis, the effect of difopein on cell death induced by antineoplastic drugs was examined. It was found that difopein enhances the ability of cisplatin to kill cells. These data support the model that 14-3-3, through binding to Bad and other ligands, is critical for cell survival signaling. Inhibition of 14-3-3 may represent a useful therapeutic target for treatment of cancer and other diseases involving inappropriate cell survival.