CD40 ligand (CD40L), a member of the tumor necrosis factor (TNF) superfamily, binds to CD40, leading to many effects depending on target cell type. Platelets express CD40L and are a major source of soluble CD40L. CD40L has been shown to potentiate platelet activation and thrombus formation, involving both CD40-dependent and -independent mechanisms. A family of proteins called TNF receptor associated factors (TRAFs) plays key roles in mediating CD40L-CD40 signaling. Platelets express several TRAFs. It has been shown that TRAF2 plays a role in CD40L-mediated platelet activation. Here we show that platelet also express TRAF3, which plays a negative role in regulating platelet activation. Thrombin- or collagen-induced platelet aggregation and secretion are increased in TRAF3 knockout mice. The expression levels of collagen receptor GPVI and integrin αIIbβ3 in platelets were not affected by deletion of TRAF3, suggesting that increased platelet activation in the TRAF3 knockout mice was not due to increased expression platelet receptors. Time to formation of thrombi in a FeCl3-induced thrombosis model was significantly shortened in the TRAF3 knockout mice. However, mouse tail-bleeding times were not affected by deletion of TRAF3. Thus, TRAF3 plays a negative role in platelet activation and in thrombus formation in vivo.