Background: T cell development results in the generation of both mature alpha beta and gamma delta T cells. While alpha beta T cells predominate in secondary lymphoid organs, gamma delta T cells are more abundant in mucosal tissues. PU.1, an Ets family transcription factor, also identified as the spleen focus forming virus proviral integration site-1 (Sfpi1) is essential for early stages of T cell development, but is down regulated during the DN T-cell stage. Methodology/Principal Findings: In this study, we show that in mice specifically lacking PU.1 in T cells using an lck-Cre transgene with a conditional Sfpi1 allele (Sfpi1(lck-/-)) there are increased numbers of gamma delta T cells in spleen, thymus and in the intestine when compared to wild-type mice. The increase in gamma delta T cell numbers in PU.1-deficient mice is consistent in gamma delta T cell subsets identified by TCR variable regions. PU.1-deficient gamma delta T cells demonstrate greater proliferation in vivo and in vitro. Conclusions/Significance: The increase of gamma delta T cell numbers in Lck-Cre deleter strains, where deletion occurs after PU.1 expression is diminished, as well as the observation that PU.1-deficient gamma delta T cells have greater proliferative responses than wild type cells, suggests that PU.1 effects are not developmental but rather at the level of homeostasis. Thus, our data shows that PU.1 has a negative influence on gamma delta T cell expansion.