We describe the development, analytical characterization, stability and preclinical efficacy of AF04, a combination adjuvant comprising the synthetic toll-like receptor 4 (TLR4) agonist, E6020, formulated in AF03, a thermoreversible squalene emulsion. By using AF04 with the recombinant major outer membrane protein of Chlamydia trachomatis (Ct-MOMP) and with the recombinant surface glycoprotein gB from human cytomegalovirus (CMV-gB) as model antigens, we show that AF03 and E6020 can synergize to augment specific antibody and Th-1 cellular immune responses in mice. In terms of formulation, we observe that the method used to incorporate E6020 into AF03 affects its partition between the oil and water phases of the emulsion which in turn has a significant impact on the tolerability (IV pyrogenicity test in rabbits) of this novel adjuvant combination.