Abstract CTLA-4 is known as a central inhibitor of T cell responses. It terminates T cell activation and proliferation and induces resistance against activation induced cell death. However, its impact on memory formation of adaptive immune responses is still unknown. In this study, we demonstrate that although anti–CTLA-4 mAb treatment during primary immunization of mice initially enhances the number of IFN-γ–producing CD4+ T cells, it does not affect the size of the memory pool. Interestingly, we find that the CTLA-4 blockade modulates the quality of the memory pool: it decreases the amount of specialized “multifunctional” memory CD4+ T cells coproducing IFN-γ, TNF-α, and IL-2 in response to Ag. The reduction of these cells causes an immense decrease of IFN-γ–producing T cells after in vivo antigenic rechallenge. Chimeric mice expressing CTLA-4–competent and –deficient cells unmask, which these CTLA-4–driven mechanisms are mediated CD4+ T cell nonautonomously. In addition, the depletion of CD25+ T cells prior to the generation of Ag-specific memory cells reveals that the constitutively CTLA-4–expressing natural regulatory T cells determine the quality of memory CD4+ T cells. Taken together, these results indicate that although the inhibitory molecule CTLA-4 damps the primary immune response, its engagement positively regulates the formation of a high-quality memory pool equipped with multifunctional CD4+ T cells capable of mounting a robust response to Ag rechallenge.