Mature dendritic cells (mDCs) can trigger the effector functions of natural killer (NK) cells. Knock-out, small-interfering RNA or neutralizing antibodies targeting interleukin 12 (IL-12) subunits revealed a critical role for IL-12 in NK cell interferon γ (IFN-γ) secretion promoted by mDCs. However, NK cell activation by DCs also required direct cell-to-cell contacts. DC-mediated NK cell activation involved the formation of stimulatory synapses between DCs and NK cells. The formation of DC/NK cell conjugates depended on cytoskeleton remodeling and lipid raft mobilization in DCs. Moreover, the disruption of the DC cytoskeleton using pharmacologic agents or the loss-of-function mutation of the Wiskott-Aldrich syndrome protein abolished the DC-mediated NK cell activation. Synapse formation promoted the polarized secretion of preassembled stores of IL-12 by DCs toward the NK cell. The synaptic delivery of IL-12 by DCs was required for IFN-γ secretion by NK cells, as assessed using inhibitors of cytoskeleton rearrangements and transwell experiments. Therefore, the cross-talk between DCs and NK cells is dictated by functional synapses. (Blood. 2004;104:3267-3275)