Accumulating evidence suggests that PD-1, an immuno-inhibitory receptor expressed on activated T cells, regulates peripheral T cell tolerance. In particular, PD-1 is involved in the induction and/or maintenance of T cells’ intrinsic unresponsiveness to previously encountered Ags, although the mechanism is yet to be determined. We used a simple experimental model to dissect the mechanism for anergy establishment, in which 2C TCR transgenic rag2−/− PD-1+/+ mice were anergized by a single injection of a cognate peptide. Interestingly, 2C rag2−/− PD-1−/− mice were totally resistant to anergy induction by the same treatment; thus, PD-1 was responsible for anergy induction. Furthermore, PD-1 expression was induced within 24 h of the initial Ag exposure. The establishment of anergy was associated with a marked down-regulation of IL-2 from the CD8+ T cells. In fact, IL-2 blockade resulted in anergy even in 2C rag2−/− PD-1−/− T cells. Furthermore, the complementation of the IL-2 signal in 2C rag2−/− PD-1+/+ mice reversed the anergy induction. We propose that CD8+ T cell anergy is induced by a reduction of cell-autonomous IL-2 synthesis, which is caused by the quick expression of PD-1 in response to Ag stimulation and the subsequent stimulation of this receptor by its ligands on surrounding cells.