Formation of migratory extravillous trophoblasts (EVTs) is critical for human placentation and hence embryonic development. However, key regulatory growth factors, hormones, and nuclear proteins controlling the particular differentiation process remain poorly understood. Here, the role of the Wingless (Wnt)-dependent transcription factor T-cell factor-4 (TCF-4) in proliferation and motility was investigated using different trophoblast cell models. Immunofluorescence of first-trimester placental tissues revealed induction of TCF-4 and nuclear recruitment of its coactivator β-catenin in nonproliferating EVTs, whereas membrane-associated β-catenin decreased upon differentiation. In addition, EVTs expressed the TCF-4/β-catenin coactivator Pygopus 2 as well as repressors of the Groucho/transducin-like enhancer of split family. Western blotting revealed Pygopus 2 expression and up-regulation of integrin α1 and nuclear TCF-4 in purified first-trimester cytotrophoblasts (CTBs) differentiating on fibronectin. Concomitantly, elevated TCF-4 mRNA, quantitated by real-time PCR, and increased TCF-dependent luciferase reporter activity were noticed in EVTs of villous explant cultures and differentiated primary CTBs. Gene silencing using specific small interfering RNA decreased TCF-4 transcript and protein levels, TCF-dependent reporter activity as well as basal and Wnt3a-stimulated migration of trophoblastic SGHPL-5 cells and primary CTBs through fibronectin-coated transwells. In contrast, proliferation of SGHPL-5 cells and primary cells, measured by cumulative cell numbers and 5-bromo-2'-deoxy-uridine labeling, respectively, was not affected. Moreover, siRNA-mediated down-regulation of TCF-4 in primary CTBs diminished markers of the differentiated EVT, such as integrin α1 and α5, Snail1, and Notch2. In summary, the data suggest that Wnt/TCF-4-dependent signaling could play a role in EVT differentiation promoting motility and expression of promigratory genes.