Tissue inflammation is characterized by increased trafficking of antigen-loaded dendritic cells (DC) from the periphery via afferent lymphatics to draining lymph nodes, with resulting stimulation of ongoing immune responses. Transmigration across lymphatic endothelium constitutes the first step in this process and is known to involve the chemokine CCL21 and its receptor CCR7. However, the precise details of DC transit remain obscure and it is likely that additional chemokine-receptor pairs have roles in lymphatic vessel entry.Here, we report that the transmembrane chemokine CX3CL1 (fractalkine) is induced in inflamed lymphatic endothelium, both in vitro in TNF-α-treated human dermal lymphatic endothelial cells (HDLEC) and in vivo in a mouse model of skin hypersensitivity. However, unlike blood endothelial cells, which express predominantly transmembrane CX3CL1 as a leukocyte adhesion molecule, HDLEC shed virtually all CX3CL1 at their basolateral surface via matrix metalloproteinases. We show for the first time that both recombinant soluble CX3CL1 and endogenous secreted CX3CL1 promote basolateral-to-luminal migration of DC across HDLEC monolayers in vitro. Furthermore, we show in vivo that neutralizing antibodies against CX3CL1 dramatically reduce allergen-induced trafficking of cutaneous DC to draining lymph nodes as assessed by FITC skin painting in mice. Finally, we show that deletion of CX3CL1 receptor in CX3CR1−/− DC results in markedly delayed lymphatic trafficking in vivo and impaired translymphatic migration in vitro, thus establishing a previously unrecognized role for this atypical chemokine in regulating DC trafficking through the lymphatics.