In the periphery, Foxp3 expression is considered sufficient to maintain natural regulatory CD4+ T-cell suppressive function. In this study, we challenge this model. Indeed, in mouse chimeras in which major histocompatibility complex (MHC) class II expression is restricted to the thymus, peripheral regulatory CD4+ T cells lack suppressive activity. In addition, regulatory CD4+ T cells recovered 5 days after transfer into recipient mice lacking expression of MHC class II molecules (self-deprived) are unable to inhibit the proliferative response of conventional CD4+ T cells both in vitro and in vivo. Disruption of TCR/MHC class II interactions rapidly leads to alterations in the regulatory CD4+ T-cell phenotype, the ability to respond to stimulation and to produce interleukin-10, and the transcriptional signature. Interestingly, self-deprivation does not affect Foxp3 expression indicating that in regulatory CD4+ T cells, self-recognition induces unique transcriptional and functional features that do not rely on Foxp3 expression.