A subset of CD8+ T cells can rapidly secrete gamma interferon (IFN-gamma) in an antigen-independent and interleukin-12 (IL-12)- and IL-18-dependent manner within 16 h of infection with the intracellular bacterial pathogen Listeria monocytogenes. This rapid IFN-gamma response is robust enough to be detected directly ex vivo and is not observed following infection with intracellular bacterial pathogens that remain sequestered within host cell vacuoles. We demonstrate here that three distinct pathways can lead to rapid secretion of IFN-gamma by CD8+ T cells during L. monocytogenes infection: (i) a direct cytokine-inducing activity encoded by the cholesterol-dependent cytolysin (CDC) listeriolysin O (LLO) acts within the infected cell, (ii) the pore-forming activity of LLO promotes cytosolic localization of bacterial products that trigger cytosol-specific signaling pathways, and (iii) the sustained presence of high concentrations of bacterial products can exogenously trigger cytokine production. Although it has been suggested that CDC protein toxins may act as Toll-like receptor 4 (TLR4) agonists to trigger proinflammatory cytokine secretion, we show in this report that TLR4 signaling is not required to induce a maximal rapid IFN-gamma response by CD8+ T cells. The results presented here indicate that multiple mechanisms contribute to the induction of rapid IFN-gamma secretion by CD8+ T cells during Listeria infection and that care must be taken when interpreting the results of in vitro assays, since the contribution of each pathway can vary depending on how the assay is performed.