CD40 ligand (CD40L) acts as an immune modulator in activated T cells, and mutations in the extracellular domain are associated to X-linked hyper IgM syndrome. A role for platelet CD40L in mediating thrombotic and inflammatory processes in atherosclerosis has also been reported. Using the Cre/loxP recombination technology we generated four knockout lines of mice with deletion of the Cd40lg gene restricted to the hematopoietic system. Mouse lines with expression of Cre recombinase driven by the Tie2, Vav1, or CD4 promoters showed in vivo ablation of CD40L in leukocytes and platelets. In contrast, in mice with Cre expression driven by the megakaryocyte lineage-restricted Pf4 promoter, abolition of CD40L expression was observed in megakaryocytes cultured in vitro, but not in circulating platelets. Characterization of these animals revealed reduced in vivo thrombogenesis and defective activation of washed CD40L-deficient platelets, suggesting that membrane-bound CD40L is involved in the control of haemostasis acting as a platelet co-activator. In addition, we report the practically absence of CD40L in mouse and human endothelial cells, as well as the detection of an exon 3-deleted CD40L transcript in both platelets and leukocytes of mouse and human origin. Finally, compared with their corresponding littermate floxed controls, Cre+ mice carrying CD40-deficient leukocytes did not exhibit increased IgM levels, and reduction of IgA and IgG levels was statistically significant only in Tie2-Cre+ mice, suggesting that expression of CD40L in an earlier developmental step may be determinant in the regulation of the class switch recombination process.