Vaccines that can rapidly induce strong and robust antibody-mediated immunity could improve protection from certain infectious diseases for which current vaccine formulations are inefficient. For indications such as anthrax and influenza, antibody production in vivo is a correlate of efficacy. Toll-like receptor (TLR) agonists are frequently studied for their role as vaccine adjuvants, largely because of their ability to enhance initiation of immune responses to antigens by activating dendritic cells. However, TLRs are also expressed on B cells and may contribute to effective B cell activation and promote differentiation into antigen-specific antibody producing plasma cells in vivo. We sought to discover an adjuvant system that could be used to augment antibody responses to influenza and anthrax vaccines. We first characterized an adjuvant system in vitro which consisted of two TLR ligands, poly I:C (TLR3) and Pam3CSK4 (TLR2), by evaluating its effects on B cell activation. Each agonist enhanced B cell activation through increased expression of surface receptors, cytokine secretion and proliferation. However, when B cells were stimulated with poly I:C and Pam3CSK4 in combination, further enhancement to cell activation was observed. Using B cells isolated from knockout mice we confirmed that poly I:C and Pam3CSK4 were signaling through TLR3 and TLR2, respectively. B cells activated with Poly I:C and Pam3CSK4 displayed enhanced capacity to stimulate allogeneic CD4+ T cell activation and differentiate into antibody-producing plasma cells in vitro. Mice vaccinated with influenza or anthrax antigens formulated with poly I:C and Pam3CSK4 in DepoVax™ vaccine platform developed a rapid and strong antigen-specific serum antibody titer that persisted for at least 12 weeks after a single immunization. These results demonstrate that combinations of TLR adjuvants promote more effective B cell activation in vitro and can be used to augment antibody responses to vaccines in vivo.