Dendritic cells (DCs) are specialized antigen-presenting cells that are present in peripheral tissues in a resting (immature) state. Their activation is a critical step in the initiation of the primary immune response. In the present study, we optimized in vitro conditions for maturation of commercially available immortalized mouse dendritic precursor JAWSII cells. These cells express surface markers and have properties that are typical of immature DCs and macrophages (e.g. MHC class I and II markers, CD80 molecules, high endocytic capacity), as well as TLR1, TLR3, TLR4, TLR6, and TLR7 receptors. When stimulated with poly I:C (and also LPS) JAWSII cells produced large amounts of IL-6, TNF-α and MCP-1. Incubation of JAWSII cells with IFN-γ markedly increased expression of MHC class I molecules and, more importantly, combination of this cytokine with poly I:C significantly increased expression of CD40 surface protein and CD11c, the most characteristic marker of mouse DCs. The combination of both agents also inhibited the endocytic abilities of JAWSII cells. In in vivo migration studies, exposure of JAWSII cells to poly I:C and IFN-γ led to increased accumulation of these cells in regional lymph nodes. Functional in vivo studies showed that tumor cell lysate-pulsed and subsequently poly I:C/IFN-γ-stimulated JAWSII cells promoted development of specific T cells in lymph nodes. Our studies show that the combination of optimal endogenous and exogenous ligands may induce phenotypic and functional maturation of JAWSII cells necessary for the accomplishment of their antigen-presenting function in vivo.