<p>Extravillous trophoblasts (EVTs) play essential roles in placental development by anchoring the placenta, invading the maternal decidua, and remodeling spiral arteries. TCF7L2 is known to be expressed in human placental tissues and EVTs, and it controls EVT motility. However, the targets of TCF7L2 in trophoblasts and the mechanism by which it contributes to early trophoblast differentiation are largely unknown. Here, using trophoblast stem cells (TSCs), we investigate the expression patterns of TCF7L2 during trophoblast lineage differentiation, revealing that its expression gradually elevates throughout EVT formation. Loss-of-function studies uncover that TCF7L2 is implicated in the proliferation of TSCs and is essential for EVT formation. Conversely, overexpression of TCF7L2 hinders TSC differentiation into STs. We identify TCF7L2 binding sites across the genome in TSCs and EVTs. Integrative analyses of TCF7L2 targets with global gene expression profiles unveil that TCF7L2 facilitates EVT formation by directly activating extracellular matrix organization while suppressing genes linked to the cell cycle. Moreover, overlap analyses of TCF7L2 targets with those of other EVT factors reveal that TCF7L2 collaborates with other EVT factors to promote EVT formation. In summary, our findings highlight context-specific functions of TCF7L2 in the trophoblast lineage.</p>