Class I PI3K enzymes are critical for the maintenance of effective immunity. In T cells, PI3Kα and PI3Kδ are activated by the T cell receptor (TCR) and costimulatory receptors, whereas PI3Kγ is activated by G protein-coupled chemokine receptors. PI3Kδ is a key regulator of regulatory T cell (Treg) function. PI3K isoform-selective inhibitors are in development for the treatment of diseases associated with immune dysregulation including chronic inflammatory conditions, cancer and autoimmune diseases. Idelalisib (PI3Kδ), Alpelisib (PI3Kα), Duvelisib (PI3Kδ/γ) and Copanlisib (pan-PI3K) have recently been approved for use in cancer treatment. Although effective, these therapies often have severe side effects associated with immune dysregulation and in particular, loss of Treg cells. Therefore, it is important to gain a better understanding of the relative contribution of different PI3K isoforms under homeostatic and inflammatory conditions. Experimental autoimmune encephalitis (EAE) is a mouse model of T cell driven central nervous system (CNS) inflammation, where Treg cells play a key protective role. Here we show that PI3Kδ is required to maintain normal Treg cell development and phenotype under homeostatic conditions, but that loss of PI3Kδ alone in Treg cells does not lead to autoimmunity. However, combined loss of PI3Kα and PI3Kδ signalling resulted in increased EAE disease severity. Moreover, mice lacking PI3Kα and PI3Kδ in Treg cells developed spontaneous peripheral nerve inflammation. These results show a key role for PI3K signalling in Treg cell-mediated protection against CNS inflammation.