Over the past 70?years, multiple approaches to develop a prophylactic or therapeutic vaccine to control herpes simplex virus (HSV) infection have failed to protect against primary infection, reactivation, or reinfection. In contrast to many RNA viruses, neither primary HSV infection nor repeated clinical recurrence elicits immune responses capable of completely preventing virus reactivation; yet the 12 known HSV-1 glycoproteins are the major inducers and targets of humoral and cell-mediated immune responses following infection. While costimulatory molecules and CD4/CD8 T cells both contribute significantly to HSV-1-induced immune responses, the specific effects of individual HSV-1 glycoproteins on CD4, CD8, CD80, and CD86 activities are not known. To determine how nine major HSV-1 glycoproteins affect T cells and costimulatory molecule function, we tested the independent effects of gB, gC, gD, gE, gG, gH, gI, gK, and gL on CD4, CD8, CD80, and CD86 promoter activities in vitro. gD, gK, and gL had a suppressive effect on CD4, CD8, CD80, and CD86 promoter activities, while gG and gH specifically suppressed CD4 promoter activity. In contrast, gB, gC, gE, and gI stimulated CD4, CD8, CD80, and CD86 promoter activities. Luminex analysis of splenocytes and bone-marrow-derived dendritic cells (BMDCs) transfected with each glycoprotein showed differing cytokine/chemokine milieus with higher responses in splenocytes than in BMDCs. Our results with the tested major HSV-1 glycoproteins suggest that costimulatory molecules and T cell responses to the nine glycoproteins can be divided into (i) stimulators (i.e., gB, gC, gE, and gI), and (ii) nonstimulators (i.e., gD, gK, and gL). Thus, consistent with our previous studies, a cocktail of select HSV-1 viral genes may induce a wider spectrum of immune responses, and thus protection, than individual genes. IMPORTANCE Currently no effective vaccine is available against herpes simplex virus (HSV) infection. Thus, there is a critical need to develop a safe and effective vaccine to prevent and control HSV infection. The development of such approaches will require an advanced understanding of viral genes. This study provides new evidence supporting an approach to maximize vaccine efficacy by using a combination of HSV genes to control HSV infection.