Engagement of TCRs induces actin rearrangements, which are critical for T cell activation. T cell responses require new actin polymerization, but the significance of higher-order actin structures, such as microfilament bundles, is unknown. To determine the role of the actin-bundling protein leukocyte-plastin (L-plastin; LPL) in this process, T cells from LPL-/- mice were studied. LPL-/- T cells were markedly defective in TCR-mediated cytokine production and proliferation. LPL-/- T cells also spread inefficiently on surfaces with immobilized TCR ligands and formed smaller immunological synapses with APCs, likely due to defective formation of lamellipodia. LPL-/- mice showed delayed rejection of skin allografts after release from immunosuppression. Moreover, LPL-/- mice developed much less severe neurologic symptoms in experimental autoimmune encephalomyelitis, which correlated with impaired T cell responses to Ag, manifested by reduced proliferation and production of IFN-g and IL-17. Thus, LPL-dependent actin bundling facilitates the formation of lamellipodia and normal immunological synapses and thereby enables T cell activation.