The placenta is a transient organ that mediates the exchange of nutrients, gases, and waste products between the mother and the developing fetus and is indispensable for a healthy pregnancy. Epithelial cells in the placenta, which are termed trophoblasts, originate from the trophectoderm (TE) compartment of the blastocyst. The human trophoblast lineage consists of several distinct cell types, including the self-renewing and bipotent cytotrophoblast and the terminally differentiated extravillous trophoblast and syncytiotrophoblast. Despite the importance of trophoblast research, it has long been hindered by the scarce accessibility of primary tissue and the lack of a robust in vitro model system. Recently, a culture condition was developed that supports the isolation of bona fide human trophoblast stem cells (hTSCs) from human blastocysts or first-trimester placental tissues. In this chapter, we describe a protocol to derive bona fide hTSCs from naïve human pluripotent stem cells (hPSCs), thus presenting a robust methodology to generate hTSCs from a renewable and widely accessible source. This approach may be used to generate patient-specific hTSCs to study trophoblast-associated pathologies and serves as a powerful experimental platform to study the specification of human TE.