The Foxp3 transcription factor is a crucial determinant of both regulatory T (TREG) cell development and their functional maintenance. Appropriate modulation of tolerogenic immune responses therefore requires the tight regulation of Foxp3 transcriptional output, and this involves both transcriptional and post-translational regulation. Here, we show that during T cell activation, phosphorylation of Foxp3 in TREG cells can be regulated by a TGF- activated kinase 1 (TAK1)-Nemo-like kinase (NLK) signaling pathway. NLK interacts and phosphorylates Foxp3 in TREG cells, resulting in the stabilization of protein levels by preventing association with the STUB1 E3-ubiquitin protein ligase. Conditional TREG cell NLK-knockout (NLKTREG) results in decreased TREG cell-mediated immunosuppression in vivo, and NLK-deficient TREG cell animals develop more severe experimental autoimmune encephalomyelitis. Our data suggest a molecular mechanism, in which stimulation of TCR-mediated signaling can induce a TAK1-NLK pathway to sustain Foxp3 transcriptional activity through the stabilization of protein levels, thereby maintaining TREG cell suppressive function. Copyright 2019 The Authors. Published by Elsevier Inc. All rights reserved.