Abstract Recent studies have shown that activation of the signaling lymphocytic activation molecule (SLAM) family of receptors plays an important role in several aspects of immune regulation. However, translation of this knowledge into a useful clinical application has not been undertaken. One important area where SLAM-mediated immune regulation may have keen importance is in the field of vaccinology. Because SLAM signaling plays such a critical role in the innate and adaptive immunity, we endeavored to develop a strategy to improve the efficacy of vaccines by incorporation of proteins known to be important in SLAM-mediated signaling. In this study, we hypothesized that coexpression of the SLAM adapter EWS-FLI1–activated transcript 2 (EAT-2) along with a pathogen-derived Ag would facilitate induction of beneficial innate immune responses, resulting in improved induction of Ag-specific adaptive immune responses. To test this hypothesis, we used rAd5 vector-based vaccines expressing murine EAT-2, or the HIV-1–derived Ag Gag. Compared with appropriate controls, rAd5 vectors expressing EAT-2 facilitated bystander activation of NK, NKT, B, and T cells early after their administration into animals. EAT-2 overexpression also augments the expression of APC (macrophages and dendritic cells) surface markers. Indeed, this multitiered activation of the innate immune system by vaccine-mediated EAT-2 expression enhanced the induction of Ag-specific cellular immune responses. Because both mice and humans express highly conserved EAT-2 adapters, our results suggest that human vaccination strategies that specifically facilitate SLAM signaling may improve vaccine potency when targeting HIV Ags specifically, as well as numerous other vaccine targets in general.