Recent studies suggest that the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) plays a critical role in the maintenance of self-tolerance. Using T cell-specific PTEN knockout mice (PTENΔT), we have identified a novel mechanism by which PTEN regulates T cell tolerance. We found that TCR stimulation alone, without CD28 costimulation, is sufficient to induce hyperactivation of the PI3K pathway, which leads to enhanced IL-2 production by naive PTENΔT T cells. Importantly, as a result of this increased response to TCR stimulation, PTENΔT CD4+ T cells no longer require CD28 costimulation for in vitro or in vivo expansion. In fact, unlike wild-type T cells, PTENΔT CD4+ T cells are not anergized by delivery of TCR stimulation alone. These data suggest that by negatively regulating TCR signals, PTEN imposes a requirement for CD28 costimulation, thus defining a novel mechanism for its role in self-tolerance.