Data have been reported on the in vivo adjuvant role of soluble lymphocyte activation gene-3 (LAG-3) recombinant protein in mouse models and on its ability to support the in vitro generation of human, tumor-specific CTLs. In this study, we show that soluble human rLAG-3 protein (hLAG-3Ig) used in vitro as a single maturation agent induces phenotypic maturation of monocyte-derived dendritic cells and promoted the production of chemokines and TNF-alpha inflammatory cytokine. When given in association with optimal or suboptimal doses of CD40/CD40L, hLAG-3Ig functions as a strong costimulatory factor and induces full functional activation of monocyte-derived dendritic cells that includes the production of high level of IL-12p70. Moreover, evidence is here provided that this costimulatory function licensing dendritic cells to produce IL-12p70 is also a functional property of LAG-3 molecules when expressed in a physiological context by CD4(+) activated T cells. Altogether, these data show for the first time a role of LAG-3 in mediating dendritic cell activation when expressed on the T cell surface or released after specific Ag stimulation in the interspaces of immunological synapses.