The ultimate goal of antitumor vaccines is to develop memory CD8(+) cytotoxic T lymphocytes (CTLs), which are critical mediators of antitumor immunity. We previously demonstrated that the ovalbumin (OVA)-specific CD4(+) T cell-based (OVA-T(EXO)) vaccine generated using OVA-pulsed dendritic cell (DC(OVA))-released exosomes (EXO(OVA)) stimulate CTL responses via IL-2 and costimulatory CD80 signaling. To assess the potential involvement of other costimulatory pathways and to define the key constituent of costimulation for memory CTL development, we first immunized wild-type (WT) C57BL/6 and gene-knockout mice with WT CD4(+) OVA-T(EXO) cells or OVA-T(EXO) cells with various molecular deficiencies. We then assessed OVA-specific primary and recall CTL responses using PE-H-2K(b)/OVA(257-264) tetramer and FITC-anti-CD8 antibody staining by flow cytometry. We also examined antitumor immunity against the OVA-expressing B16 melanoma cell line BL6-10(OVA). We demonstrated that CD4(+) OVA-T(EXO) cells stimulated more efficient CTL responses compared to DC(OVA). By assessing primary and recall CTL responses in mice immunized with OVA-T(EXO) or with OVA-T(EXO) lacking the costimulatory molecules CD40L, 4-1BBL or OX40L, we demonstrated that these costimulatory signals are dispensable for CTL priming by OVA-T(EXO). Interestingly, CD40L, but not 4-1BBL or OX40L, plays a crucial role in the development of functional memory CTLs against BL6-10(OVA) tumors. Overall, this work suggests that a novel CD4(+) T cell-based vaccine that is capable of stimulating long-term functional CTL memory via CD40L signaling may represent a novel, efficient approach to antitumor vaccination.