The importance of CLEC-2, a natural ligand/receptor for Gp38/Podoplanin, in the formation of the lymphatic vasculature has recently been demonstrated. As the development and maintenance of lymph nodes is dependent on the formation of the lymphatic vasculature and the differentiation of a Gp38/Podoplanin(+) stromal cells, we investigated the role of CLEC-2 in lymphoneogenesis and lymph node homeostasis. Using constitutive Clec1b(-/-) mice, we showed that while CLEC-2 was not necessary for initiation of the lymph node anlage, it was required at late stages of development. Constitutive deletion of CLEC-2 induced a profound defect in lymphatic endothelial cell proliferation, resulting in lack of lymph nodes at birth. In contrast, conditional deletion of CLEC-2 in the megakaryocyte/platelet lineage in Clec1b(fl/fl)PF4-Cre mice led to the development of blood-filled lymph nodes and fibrosis, in absence of a proliferative defect of the lymphatic endothelial compartment. This phenotype was also observed in chimeric mice reconstituted with Clec1b(fl/fl)PF4-Cre bone marrow, indicating that CLEC-2 expression in platelets was required for lymph node integrity. We demonstrated that lymph nodes of Clec1b(fl/fl)PF4-Cre mice are able to sustain primary immune responses but show a defect in immune cell recirculation after repeated immunizations thus suggesting CLEC-2 as target in chronic immune response.