T cell homeostasis is crucial for maintaining an efficient and balanced T cell immunity. The interaction between TCR and self peptide (sp) MHC ligands is known to be the key driving force in this process, and it is believed to be functionally and mechanistically different from that initiated by the antigenic TCR stimulation. Yet, very little is known about the downstream signaling events triggered by this TCR-spMHC interaction and how they differ from those triggered by antigenic TCR stimulation. In this study, we show that T cell conditional ablation of MEKK3, a Ser/Thr kinase in the MAPK cascade, causes a significant reduction in peripheral T cell numbers in the conditional knockout mice, but does not perturb thymic T cell development and maturation. Using an adoptive mixed transfer method, we show that MEKK3-deficient T cells are severely impaired in lymphopenia-induced cell proliferation and survival. Interestingly, the Ag-induced T cell proliferation proceeds normally in the absence of MEKK3. Finally, we found that the activity of ERK1/2, but not p38 MAPK, was attenuated during the lymphopenia-driven response in MEKK3-deficient T cells. Together, these data suggest that MEKK3 may play a crucial selective role for spMHC-mediated T cell homeostasis.