This study describes the generation of trophoblast organoids using human trophoblast stem (TS) cells, which can form spherical structures with a single layer of syncytiotrophoblast (ST) cells that exhibit barrier function. The authors developed a column-type ST barrier model based on the culture conditions of these organoids, where the bottom membrane is almost entirely covered with ST cells. The integrity and maturation of the model were confirmed by measuring transepithelial/transendothelial electrical resistance (TEER) and the amount of human chorionic gonadotropin (hCG). The model was also used to derive apparent permeability coefficients of model compounds, demonstrating its utility in studying placental development and compound transfer and toxicity. The trophoblast models provide a valuable tool for evaluating compound transfer and toxicity, facilitating drug development and advancing our understanding of placental physiology and pathology.This study describes the generation of trophoblast organoids using human trophoblast stem (TS) cells, which can form spherical structures with a single layer of syncytiotrophoblast (ST) cells that exhibit barrier function. The authors developed a column-type ST barrier model based on the culture conditions of these organoids, where the bottom membrane is almost entirely covered with ST cells. The integrity and maturation of the model were confirmed by measuring transepithelial/transendothelial electrical resistance (TEER) and the amount of human chorionic gonadotropin (hCG). The model was also used to derive apparent permeability coefficients of model compounds, demonstrating its utility in studying placental development and compound transfer and toxicity. The trophoblast models provide a valuable tool for evaluating compound transfer and toxicity, facilitating drug development and advancing our understanding of placental physiology and pathology.