Cellular fibronectin containing extra domain A (Fn-EDA+), which is produced in response to tissue injury in several disease states, has prothrombotic activity and is known to interact with Toll-like-receptor 4 (TLR4). The underlying mechanism and cell types involved in mediating the prothrombotic effect of Fn-EDA+ still remain unknown. Using intravital microscopy, we evaluated susceptibility to carotid artery thrombosis after FeCl₃-induced injury in mice expressing Fn lacking EDA (Fn-EDA^−/− mice) or Fn containing EDA (Fn-EDA^+/+ mice). Fn-EDA^−/− mice exhibited prolonged times to first thrombus formation and complete occlusion and a significant decrease in the rate of thrombus growth (P < .05 vs Fn-EDA^+/+ mice). Genetic deletion of TLR4 reversed the accelerated thrombosis in Fn-EDA^+/+ mice (P < .05) but had no effect in Fn-EDA^−/− mice. Bone marrow transplantation experiments revealed that TLR4 expressed on hematopoietic cells contributes to accelerated thrombosis in Fn-EDA^+/+ mice. In vitro studies showed that cellular Fn-EDA+ interacts with platelet TLR4 and promotes agonist-induced platelet aggregation. Finally, Fn-EDA^+/+ mice specifically lacking platelet TLR4 exhibited prolonged times to first thrombus formation and complete occlusion (P < .05 vs Fn-EDA^+/+ mice containing platelet TLR4). We conclude that platelet TLR4 contributes to the prothrombotic effect of cellular Fn-EDA+, suggesting another link between thrombosis and innate immunity.