The human placenta provides the site of exchange between the maternal and fetal bloodstreams, acts as an endocrine organ, and has immunological functions. The majority of pregnancy disorders including preeclampsia and fetal growth restriction have their roots in pathological placentation. Yet, the underlying molecular causes of these complications remain largely unknown, not least due to the lack of reliable in vitro models. Recent establishment of 2D human trophoblast stem cells and 3D trophoblast organoids has been a major advancement that opened new avenues for trophoblast research. Here we provide a protocol detailing isolation of cytotrophoblast from the first trimester human placenta, establishment of trophoblast organoids, their culture and differentiation conditions. Overall, we describe an in vitro system that offers an excellent model to study the molecular basis of placental development and disease.