Dysregulated generation and/or function of naturally occurring 'CD4(+)CD25(+) regulatory T cells' (T(reg)s) play key role in the development of autoimmune diseases, including type 1 diabetes. Recent findings suggest that Notch3 signaling activation promotes thymic generation and peripheral expansion and in vivo function of naturally occurring T(reg)s, thus preventing autoimmune diabetes progression in mouse models. However, the mechanisms underlying these effects have remained elusive, thus far. Here, we show that the expression of pTalpha gene is up-regulated in naturally occurring T(reg)s, at both mRNA and protein levels. Moreover, by using double mutant mice, with T cell-targeted constitutive activation of Notch3 in a pTalpha(-/-) background, we demonstrate that pTalpha deletion significantly counteracts the Notch3-dependent expansion, the increased FoxP3 expression and the enhanced in vitro activity of naturally occurring T(reg)s. Notably, the absence of pTalpha also impairs the Notch3-dependent protection against experimentally induced autoimmune diabetes. Finally, by adoptive cell transfer experiments, we demonstrated that this failure is directly related to the impaired in vivo function of naturally occurring T(reg)s bearing pTalpha deletion. Collectively, our data suggest that pTalpha expression is required for the in vivo function of naturally occurring T(reg)s and that the activation of Notch3 signaling may positively regulate the function of this population, through the pTalpha/pre-T cell receptor pathway.