It has been suggested that high affinity/avidity interactions are required for the thymic selection of Treg. Here, we investigated the role of CD5, a negative regulator of TCR signaling, in the selection and function of "naturally occurring" CD4+CD25+ Treg (nTreg). Analysis of CD5-/- mice showed a significant increase in the percentage and absolute numbers of CD4+ CD25+Foxp3 + thymocytes and peripheral T lymphocytes, compared with BALB/c mice. Thymi from CD5-/- mice showed reduced cellularity due to increased apoptosis, which preferentially affected naïve T cells. To characterize nTreg selection at themolecular level we investigated the phosphorylation of Erk, c-Cbl, PI3K and Akt. CD5-/- nTreg showed increased basal levels of p-Erk compared with wild-type nTreg. Interestingly, in response to CD3 plus CD28 costimulation, CD5-/- naïve T cells but not CD5 -/- nTreg showed lower levels of p-Akt. Finally, CD5-/- nTreg were thymus-derived and fully functional. We conclude that the enrichment of nTreg observed in the absence of CD5 signaling is due to de novo generation of nTreg and selective reduction of CD4+CD25- naïve thymocytes. Furthermore, we provide new evidence supporting a potential role of CD5 in thymocyte survival, through a mechanism that may involve the phosphorylation of Akt. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA.