The p38 mitogen-activated protein kinase (p38MAPK) is activated in response to various stimuli, including cellular stress, inflammatory cytokines and cell surface receptors. The activation of p38MAPK is predominantly mediated by the two upstream MAPK kinases MKK3 and MKK6. To study the role of the p38MAPK pathway in vivo, we generated Mkk6−/− mice. We examined whether T-cell apoptosis is affected in these mice and in our previously reported Mkk3−/− mice. Strikingly, in vivo deletion of double positive thymocytes in Mkk6−/− mice was impaired, whereas Mkk3−/− mice showed no apparent abnormality. Conversely, CD4+ T cells from Mkk3−/− but not from Mkk6−/− mice were resistant to activation-induced cell death and cytokine-withdrawal-induced apoptosis. In peripheral CD4+ T cells, MKK3 is induced upon stimulation, whereas MKK6 is downregulated. These results suggest a novel mechanism regulating T-cell apoptosis differentially through the p38MAPK pathway by MKK3 and MKK6.