Regulatory T cells (Tregs) are critical for actively maintaining immune tolerance. The Foxp family of transcription factors is composed of four members; Foxp1, Foxp3 and Foxp4 are expressed in lymphocytes. Foxp family members bind DNA as homo- and hetero-dimers to regulate gene expression. Regulatory T cells are CD4+ T cells that express Foxp3. We have generated mice in which both Foxp1 and Foxp4 are deleted in either all T lymphocytes or in Tregs, leaving Foxp3 the only potentially expressed family member in this T cell subset. Our preliminary data demonstrate that combined loss of Foxp1 and Foxp4 in all T lymphocytes based on CD4 Cre-mediated deletion substantially alters the development of Tregs in the thymus, reduces peripheral Treg cellularity, and alters suppressive function. When loss of Foxp1 and Foxp4 is limited to the Foxp3+ Treg population, there is a dramatic phenotype characterized by lymphocyte activation/expansion, autoantibody production, and early lethality. The overall goal of this proposal is to understand how Foxp1 and Foxp4 alter the development, homeostasis and function of Foxp3+ Tregs. To address this question we propose to generate and use novel mouse strains in which Foxp1 and Foxp4 are deleted at different stages of Treg development. We will use high throughput sequencing technology to investigate gene regulation by Foxp1 and/or Foxp4. We will test Tregs deficient in Foxp1 and Foxp4 in relevant models of autoimmunity and transplantation. Understanding these fundamental aspects of Treg generation, homeostasis and function are critical to translational strategies of Treg augmentation being developed for use in autoimmune disease and solid-organ transplantation.