Cbl-b(-/-) mice have signaling defects that result in CD28-independent T cell activation, increased IL-2 production, hyper-reactive T cells, and increased autoimmunity. Although the increased autoimmunity in these mice is believed to result from the hyper-reactive T cells, the mechanisms leading from T cell hyper-reactivity to autoimmunity remain unclear. Specifically, the function and interaction of CD4(+)CD25(+) regulatory T cells (T(reg)) and CD4(+)CD25(-) effector T cells (T(eff)) in Cbl-b(-/-) mice have not been examined. We now report that Cbl-b(-/-) CD4(+)CD25(+) T(reg) exhibit normal regulatory function in vitro. In contrast, the in vitro response of Cbl-b(-/-) CD4(+)CD25(-) T(eff) is abnormal, in that it is not inhibited by either Cbl-b(-/-) or wild-type T(reg). This resistance of Cbl-b(-/-) T(eff) to in vitro regulation is seen at the levels of both DNA synthesis and cell division. In addition to this resistance to CD4(+)CD25(+) T(reg), Cbl-b(-/-) T(eff) demonstrate in vitro resistance to inhibition by TGF-beta. This second form of resistance in Cbl-b(-/-) T(eff) is seen despite the expression of normal levels of type II TGF-beta receptors and normal levels of phosphorylated Smad3 after TGF-beta stimulation. Coupled with recent reports of resistance to T(reg) in T(eff) exposed to LPS-treated dendritic cells, our present findings suggest that resistance to regulation may be a relevant mechanism in both normal immune function and autoimmunity.