Objective We investigated the hypothesis that antiphospholipid antibodies (aPL) have a detrimental effect on human extravillous trophoblast (EVT) differentiation into giant multinucleated cells “in vitro.” Design The EVT were isolated from the placental chorion using enzymatic digestion and Percoll gradient centrifugation. After 24, 36, and 48 hours in culture, giant multinuclear cells (GMC) were identified by immunohistochemistry using antibodies to cytokeratin 7 and counted. Setting An academic research laboratory. Patient(s) Placentas were donated by women having an elective cesarean section for a normal pregnancy at term. Main outcome measure(s) This model was then used to investigate the effect of two different monoclonal aPL to β2-glycoprotein 1 (IIC5 and ID2), and control mouse IgG antibody on EVT differentiation. Result(s) Freshly isolated EVT were nonproliferative but moved together losing their intervening cell walls and differentiated into GMC. Maximal numbers of GMC were detected after 48 hours of culture. The aPL, IIC5, and ID2 significantly inhibited GMC formation, whereas the mouse IgG control had no effect. Conclusion(s) Antiphospholipid antibodies can inhibit EVT differentiation and GMC formation “in vitro” suggesting that a failure of trophoblast differentiation and subsequent uteroplacental development may be an underlying pathology in antiphospholipid syndrome-associated pregnancy loss.