Cytotoxic T cell (CTL) is the key to induce an effective immune response against infections caused by intracellular pathogens, such as virus and cancer. CTLs with low affinity can induce and maintain a substantial population during an adaptive immune response, although CTLs with a highest-affinity receive competitive activation signals. Low-affinity CTLs are important to induce effector response and maintain a diverse memory repertoire. However, the mechanism of generating and maintaining the expansion of lower affinity CTLs is still unknown. Here, we showed that the presence of exosome (Exo) enhanced the CTL survival and increased the cell proliferation especially in CTLs treated with the low-affinity peptide. Exo together with peptides also improved the production of IFN-γ and GZB. The exosomal stimulation in CTLs was relative to up-regulation of CD25 expression, which enhanced the IL-2 survival signals. Moreover, Exo derived from an early stage of activation had a similar but weaker function comparing with Exo derived from a late stage of activation in activating CTLs. This study identified the important role of the exosome derived from CTLs stimulated by the highest-affinity peptide in activating the naïve T cells stimulated by the low-affinity peptide.