At the first cell fate decision in mammalian development, the origins of trophoblast and embryonic cell lineages are established as the trophectoderm and the inner cell mass (ICM) in the blastocyst. In the trophoblast cell lineage, a subset of the trophectoderm cells maintains the capacity to proliferate and contribute to the extraembryonic ectoderm, the ectoplacental cone, and the secondary giant cells of the early conceptus after implantation, and finally they produce the entire trophoblastic population in the mature placenta. The stem cell population of the trophectoderm lineage can be isolated and maintained in vitro in the presence of fibroblast growth factor 4, heparin, and a feeder layer of mouse embryonic fibroblast cells. These apparently immortal stem cells in culture are termed trophoblast stem (TS) cells, and exhibit the potential to differentiate into multiple trophoblastic cell types in vitro, as well as in vivo. Even after multiple passages, TS cells retain the ability to participate in the normal development of chimeras and contribute exclusively to the trophoblastic component of the placenta and of the parietal yolk sac. The fate of TS cells is strikingly in contrast to that of embryonic stem cells, which never contribute to these tissues. In this chapter, detailed protocols for the isolation and establishment of TS cell lines from blastocysts and their maintenance are described.