Impaired immune responsiveness is a significant barrier to vaccination of neonates. By way of example, the low seroconversion observed following influenza vaccination has led to restriction of its use to infants over 6 months of age, leaving younger infants vulnerable to infection. Our previous studies using a non‐human primate neonate model demonstrated that the immune response elicited following vaccination with inactivated influenza virus could be robustly increased by inclusion of the Toll‐like receptor agonist flagellin or R848, either delivered individually or in combination. When delivered individually, R848 was found to be the more effective of the two. To gain insights into the mechanism through which these adjuvants functioned in vivo, we assessed the initiation of the immune response, i.e. at 24 hr, in the draining lymph node of neonate non‐human primates. Significant up‐regulation of co‐stimulatory molecules on dendritic cells could be detected, but only when both adjuvants were present. In contrast, R848 alone could increase the number of cells in the lymph node, presumably through enhanced recruitment, as well as B‐cell activation at this early time‐point. These changes were not observed with flagellin and the dual adjuvanted vaccine did not promote increases beyond those observed with R848 alone. In vitro studies showed that R848 could promote B‐cell activation, supporting a model wherein a direct effect on neonate B‐cell activation is an important component of the in vivo potency of R848 in neonates.