Antiphospholipid Abs (APLAs) are associated with thrombosis and recurrent fetal loss. These Abs are primarily directed against phospholipid-binding proteins, particularly β₂GPI, and activate endothelial cells (ECs) in a β₂GPI-dependent manner after binding of β₂GPI to EC annexin A2. Because annexin A2 is not a transmembrane protein, the mechanisms of APLA/anti-β₂GPI Ab–mediated EC activation are uncertain, although a role for a TLR4/myeloid differentiation factor 88–dependent pathway leading to activation of NF-κB has been proposed. In the present study, we confirm a critical role for TLR4 in anti-β₂GPI Ab–mediated EC activation and demonstrate that signaling through TLR4 is mediated through the assembly of a multiprotein signaling complex on the EC surface that includes annexin A2, TLR4, calreticulin, and nucleolin. An essential role for each of these proteins in cell activation is suggested by the fact that inhibiting the expression of each using specific siRNAs blocked EC activation mediated by APLAs/anti-β₂GPI Abs. These results provide new evidence for novel protein-protein interactions on ECs that may contribute to EC activation and the pathogenesis of APLA/anti-β₂GPI–associated thrombosis and suggest potential new targets for therapeutic intervention in antiphospholipid syndrome.