We conducted a study to evaluate the protective efficacy in mice of vaccination with novel adenovirus vectors expressing an influenza A nucleoprotein (AdFluA-NP) based on isolates from non-human primates. In a previous study, we had observed that AdFluA-NP vectors can induce similar T cell responses in mice yet differ in ability to protect animals from lethal challenge with influenza A virus. To better define correlates of protection, we extended our study design to include additional novel AdFluA-NP vectors, and to evaluate cytotoxic T lymphocyte (CTL) responses in the spleens and lungs of immunized mice prior to virus challenge. As in our previous study, all vectors induced similar numbers of antigen-specific interferon gamma (IFNgamma) secreting T cells and memory T cells in the spleen 4 weeks post immunization, but differed in their ability to protect the animals from lethal infection. However, cytokine-secreting NP antigen-specific CTLs in the lungs of mice from immunization groups that survived lethal challenge showed greater proliferative ability and higher CD27 expression. In addition, NP antigen-specific peripheral blood lymphocytes from protected mice showed greater proliferative ability after ex vivo stimulation. Our results provide additional correlates of protection that should be considered when developing anti-influenza vaccines.