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Trophoblast stem cells, derived from the trophectoderm of the blastocyst, are used as an in vitro model to reveal the mechanisms underlying placentation in mammals. In humans, suitable culture conditions for trophoblast stem cell derivation have recently been established. The established human trophoblast stem cells differentiate efficiently toward two trophoblast subtypes: syncytiotrophoblasts and extravillous trophoblasts. However, the efficiency of differentiation is lower in macaque trophoblast stem cells than in human trophoblast stem cells. Here, we demonstrate that the activation of Wnt signaling downregulated the expression of inhibitory G protein and induced trophoblastic lineage switching to the syncytiotrophoblast progenitor state. The treatment of macaque trophoblast stem cells with a GSK-3 inhibitor, CHIR99021, upregulated syncytiotrophoblast progenitor markers and enhanced proliferation. Under the Wnt signaling–activated conditions, macaque trophoblast stem cells effectively differentiated to syncytiotrophoblasts upon dibutyryl cyclic AMP (dbcAMP) and forskolin treatment. RNA-seq analyses revealed the downregulation of inhibitory G protein, which may make macaque trophoblast stem cells responsive to forskolin. Interestingly, this lineage switching appeared to be reversible as the macaque trophoblast stem cells lost responsiveness to forskolin upon the removal of CHIR99021. The ability to regulate the direction of macaque trophoblast stem cell differentiation would be advantageous in elucidating the mechanisms underlying placentation in non-human primates.The activation of Wnt signaling in macaque trophoblast stem cell lines potentiated differentiation toward syncytiotrophoblast cell fate.Graphical Abstract