MF59 is a safe and effective vaccine adjuvant that has been used in a licensed seasonal influenza vaccine for 15 years. The purpose of the present studies was to directly address a question that has been asked of us on many occasions: “which is the adjuvant active component of MF59?”. Since we have recently gained a number of insights on how MF59 works as an adjuvant, we were able to use these approaches to evaluate if the individual components of MF59 (squalene oil, the surfactants Span 85 and Tween 80 or the citrate buffer) showed any direct immunostimulatory activity. We assessed the ability of the individual components to stimulate the innate and adaptive immune responses that we have shown to be indicative of MF59-mediated adjuvanticity. No immune stimulatory capacities could be attributed to squalene, Tween 80 or the citrate buffer alone. Instead, we found that the lipophilic surfactant Span 85 contributes to activation of the muscle transcriptome. However, despite this local activation, Span 85 alone – like the other single components of MF59 – is not sufficient to induce an adjuvant effect. Only the fully formulated MF59 emulsion induces all the established hallmarks of innate and adaptive immune activation, which includes activation of genes indicative of transendothelial cell migration, strong influx of immune cells into the injection site and their enhanced antigen uptake and transport to the lymph nodes. These observations may have important implications in the design of optimal emulsion-based vaccine adjuvants.