Exposure of naive CD8 T cells to the synergistic combination of interleukin (IL)-7 and IL-21 enables them to respond strongly to subsequent antigen stimulation. Mechanisms underlying the increased antigen responsiveness of such cytokine-primed CD8 T cells remain unknown. In this study, we showed that a brief exposure of <24 h to IL-7 and IL-21 is sufficient enough to sensitize naive P14 T-cell receptor (TCR) transgenic CD8 T cells to respond to limiting quantities of antigen, resulting in increased proliferation, interferon-gamma secretion and antigen-specific cytolytic activity. Cytokine-induced increase in TCR responsiveness occurs even in the absence of costimulatory signals. Cytokine priming upregulates the expression of the gamma(c) chain and increases IL-2 production after antigen stimulation, thus enhancing autocrine stimulation. Notably, cytokine priming induces a rapid and profound downmodulation of CD5, implicated in the negative regulation of TCR signaling, by induction of the transcriptional repressor E47. These findings show that increased antigen responsiveness of cytokine-primed CD8 T cells results from the modulation of multiple cell-surface molecules, which influence cytokine receptor and TCR signaling.