Differentiation of extravillous trophoblast cells (EVT) to an invasive phenotype plays an essential role in establishing and maintaining feto-placental organization during human pregnancy. A switch in integrin expression occurs during this differentiation and is accompanied by changes in the extracellular matrix (ECM). Alteration of EVT behavior is also modulated by cytokines. To investigate the molecular interactions involved in the EVT differentiation, we examined the effects of cytokines and ECM on the human EVT cell line, TCL1 cells. We found that tumor necrosis factor alpha (TNFα) induced apoptosis in TCL1 cells but not in JEG3 cells derived from choriocarcinoma while the addition of interleukin-1β, leukemia inhibitory factor, or transforming growth factor had no effect on TCL1 cells. This apoptosis was suppressed when TCL1 cells were seeded on fibronectin (Fn), collagen type I (C1), collagen type IV (C4), or laminin (Ln). Wortmannin, a specific PI3 kinase inhibitor, inhibited this suppression. Spreading assays and adhesion blocking assays indicated that TCL1 cells express integrin-α5 and -α6 and β1 and β4 subunits. Adhesion on Fn is mediated by α5β1, and adhesion on C1, C4, or Ln is mediated by α6β1 integrins. TNFα suppressed α6 integrin expression and enhanced α1 integrin expression in a dose-dependent manner. In addition, aggregation of β1 subunits on C4 was detected after addition of TNFα. Taken together, these results suggest that TNFα and ECM, through activation of PI3 kinase mediated by β1 integrin signaling, might collaboratively regulate differentiation of trophoblast cells through integrin signaling in establishing and maintaining successful pregnancy.