A combination of the pro-inflammatory cytokines interleukin (IL)-1α, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α induces nitric oxide synthase mRNA expression and nitric oxide (NO) generation in the human colon carcinoma cell line HT-29. This can be inhibited by pretreatment with IL-13 via a phosphatidylinositol (PI) 3-kinase-dependent mechanism (Wright, K., Ward, S. G., Kolios, G., and Westwick, J. (1997)J. Biol. Chem. 272, 12626–12633). Since NO has been implicated in regulating mechanisms leading to cell death, while activation of PI 3-kinase-dependent signaling cascades are thought to be involved with promoting cell survival events, we have investigated the outcome of these cytokine treatments on apoptosis and cell survival of HT-29 cells. Initiation of apoptosis can be achieved by the combinations of IFN-γ/TNF-α, IFN-γ/CD95, IL-1α/IFN-γ, and IL-1α/IFN-γ/TNF-α to varying extents. Induction of apoptotic markers by HT-29 cells in response to cytokine treatment is not dependent on NO production. Pretreatment with IL-13 protects against IL-1α/IFN-γ/TNF-α- and IFN-γ/TNF-α- as well as IFN-γ/CD95-induced (but not IL-1α/IFN-γ-induced) cell death. In addition, IFN-γ/TNF-α and IL-1α/IFN-γ/TNF-α stimulate activation of caspase-8 and caspase-3, which IL-13 pretreatment was able to partially inhibit and delay. IL-13 also stimulates activation of the major PI 3-kinase effector, protein kinase B. The PI 3-kinase inhibitors wortmannin and LY294002 inhibit IL-13 stimulation of protein kinase B as well as the cell survival effects of IL-13. These data demonstrate that cytokine-induced apoptosis of HT-29 cells is NO-independent and that the activation of a PI 3-kinase-dependent signaling cascade by IL-13 is a key signal responsible for the inhibition of apoptosis.