Cytokines such as IL-1α, IL-1β, and IFN-γ have long been implicated in the pathogenesis of autoimmune diabetes, but the mechanisms through which they promote diabetogenesis remain unclear. Here we show that CD4⁺ T lymphocytes propagated from transgenic nonobese diabetic (NOD) mice expressing the highly diabetogenic, β cell–specific 4.1-T-cell receptor (4.1-TCR) can kill IL-1α–, IL-1β–, and IFN-γ–treated β cells from NOD mice. Untreated NOD β cells and cytokine-treated β cells from Fas-deficient NOD.lpr mice are not targeted by these T cells. Killing of islet cells in vitro was associated with cytokine-induced upregulation of Fas on islet cells and was independent of MHC class II expression. Abrogation of Fas expression in 4.1-TCR–transgenic NOD mice afforded nearly complete protection from diabetes and did not interfere with the development of the transgenic CD4⁺ T cells or with their ability to cause insulitis. In contrast, abrogation of perforin expression did not affect β cell–specific cytotoxicity or the diabetogenic potential of these T cells. These data demonstrate a novel mechanism of action of IL-1α, IL-1β, and IFN-γ in autoimmune diabetes, whereby these cytokines mark β cells for Fas-dependent lysis by autoreactive CD4⁺ T cells.