During immune responses, activated endothelial cells down-regulate thrombomodulin and up-regulate tissue factor expression leading to the development of a procoagulant surface. CD4+ T cells are known to promote endothelial cell procoagulant activity, however, the molecular interactions that mediate this effect are not completely known. CD40L is an activation-induced CD4+ T cell surface molecule that functionally interacts with CD40 expressed on endothelial cells. In this study we ask if CD40L-CD40 interactions modulate endothelial cell surface tissue factor or thrombomodulin expression in vitro. Human umbilical vein endothelial cells (HUVEC) were cocultured with control cells or CD40L+ Jurkat T cells in the presence or absence of anti-CD40L mAb. By two-color FACS analysis we demonstrated that CD40 ligation induces HUVEC tissue factor expression and thrombomodulin down-regulation. Utilizing neutralizing antibodies, we show that CD40L-mediated tissue factor and thrombomodulin modulation, as well as E-selectin and VCAM-1 up-regulation, is independent of tumor necrosis factor α, interleukin-1α, or interleukin-1β production. Together these data suggest that CD40L-CD40 interactions may directly regulate endothelial cell procoagulant activity during inflammatory responses. J. Leukoc. Biol. 63: 373–379; 1998.