Dendritic cells (DCs) produce major histocompatibility complex II (MHC II) in large amounts to function as professional antigen presenting cells. Paradoxically, DCs also ubiquitinate and degrade MHC II in a constitutive manner. Mice deficient in the MHC II-ubiquitinating enzyme membrane-anchored RING-CH1, or the ubiquitin-acceptor lysine of MHC II, exhibit a substantial reduction in the number of regulatory T (Treg) cells, but the underlying mechanism was unclear. Here we report that ubiquitin-dependent MHC II turnover is critical to maintain homeostasis of lipid rafts and the tetraspanin web in DCs. Lack of MHC II ubiquitination results in the accumulation of excessive quantities of MHC II in the plasma membrane, and the resulting disruption to lipid rafts and the tetraspanin web leads to significant impairment in the ability of DCs to engage and activate thymocytes for Treg cell differentiation. Thus, ubiquitin-dependent MHC II turnover represents a novel quality-control mechanism by which DCs maintain homeostasis of membrane domains that support DC's Treg cell-selecting function.