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-β. This second form of resistance in Cbl-b−/− T eff is seen despite the expression of normal levels of type II TGF-β receptors and normal levels of phosphorylated Smad3 after TGF-β 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.