The immune system protects itself from autoreactivity by maintaining a balance between effector and Treg responses. Peripheral induction of Treg is one mechanism by which this balance may be maintained. Thus, it is important to understand factors that influence de novo generation of CD4(+)CD25(+)FOXP3(+) Treg. Here, we focus on the effects of cytokines and the cell cycle inhibitor rapamycin. The cytokines IL-2 and IL-7, but not IL-4, increased initial activation induced FOXP3 expression, increased proliferation and sustained expression of FOXP3(+) cells throughout the culture. Addition of rapamycin to cultures containing IL-2 further increased the frequency and absolute number of functional CD4(+)CD25(+)FOXP3(+) Treg. This increase was not due to selective proliferation of FOXP3 cells, but was instead, the result of an increase in the frequency of FOXP3(+) cells induced in G0 through delayed activation while the addition of IL-2 promoted survival and proliferation of the FOXP3(+) population. Thus, combination of rapamycin and IL-2 may provide improved treatment options in transplantation and autoimmunity by promoting induction, survival, and expansion of functional iTreg from CD4(+)CD25(-) cells.