AbstractStimulation via the T-cell receptor (TCR) activates p38α and p38β by phosphorylation of p38 Tyr-323 (p38Y323). Here we characterize knockin mice in which p38α and/or β Tyr-323 has been replaced with Phe. We find that p38α accounts for two-thirds and p38β the remainder of TCR-induced p38 activation. T cells from double knockin mice (p38αβY323F) had defects in TCR-mediated proliferation and Th1 and Th17 skewing, the former corresponding with an inability to sustain T-bet expression. Introduction of p38αY323F into Gadd45α-deficient mice, in which the alternative p38 pathway is constitutively active, reversed T-cell hyperproliferation and autoimmunity. Furthermore, p38αβY323F mice had delayed onset and reduced severity of the inflammatory autoimmune diseases collagen-induced arthritis and experimental autoimmune encephalomyelitis. Thus, T cell-specific alternative activation of p38 is an important pathway in T-cell proliferation, Th skewing, and inflammatory autoimmunity, and may be an attractive tissue-specific target for intervention in these processes.