Regulatory T cell (Treg) development proceeds via a two-step process in which naive CD4+ thymocytes are first converted into CD4 +CD25+CD122+GITR+Foxp3- Treg progenitors, followed by a second step in which IL-2 converts these Treg progenitors into CD4+Foxp3+ Tregs. The costimulatory molecule CD28 is required for efficient Treg development. However, the stage at which CD28 affects Treg development remains undefined. In this article, we demonstrate that Cd28-/- mice lack Treg progenitors. Furthermore, the P187YAP motif in the cytoplasmic tail of CD28, which links CD28 to Lck activation, is required for this process. In contrast, the Y 170MNM motif, which links CD28 to PI3K activation, is not required for Treg progenitor development. Finally, the CD28/Lck pathway was shown to activate the NF-κB family of transcription factors. We demonstrate that c-Rel, but not NF-κB1, promotes the development of Treg progenitors. Thus, a CD28/c-Rel-dependent pathway is involved in initiating Treg development.