Regulatory T (Treg) cells play an indispensable role in maintaining immune homeostasis and preserving tolerance to self and innocuous antigens, however they can also inhibit anti-tumour immunity. Treg cells are defined by the expression of the master transcription factor FOXP3, and can develop during negative selection in the thymus (tTreg) or be induced (iTreg) from naive CD4+ T cells in vivo or in vitro in the presence of IL-2 and TGF-β. Along with the establishment of a specific transcriptional program, epigenetic regulation has also been shown to be critical for the development, stability and function of Treg cells. We have previously identified the lysine methyltransferase DOT1L as a key regulator of CD4+ T helper (Th) cell lineage integrity and function [1]. Here, we show that DOT1L is also a novel regulator of Treg cell development, stability and function. The T cell intrinsic deletion of DOT1L led to a significant loss of mature Treg cells in the thymus, secondary lymphoid and non-lymphoid tissues. In the absence of DOT1L, iTreg cell development was not only impaired but also highly dysregulated, resulting in FOXP3 expressing iTreg cells producing the proinflammatory cytokine IFN-g. Furthermore, ATAC-sequencing of DOT1L-defiicient FOXP3+ iTreg cells revealed a dramatic reduction in chromatin accessibility at the Foxp3 promoter and conserved non-coding regions, yet a gain of accessibility at Th1 associated genes Tbx21 and Ifng. Strikingly, Treg-specific ablation of DOT1L resulted in severe spontaneous autoimmune and inflammatory disease at 3-4 weeks of age. Interestingly, FOXP3+ Treg cells were still present in lymphoid tissues, further highlighting DOT1L as a critical factor in maintaining the function and identity of Treg cells. Taken together, these findings identify DOT1L as a novel regulator of Treg cell development, stability and function, potentially serving as a target for destabilising Treg cells in disease settings such as cancer.