The trophectoderm is one of the earliest cell types to differentiate in the forming mammalian embryo. It is responsible for the initial implantation and the formation of the trophoblast components of the placenta, an organ essential for nutrient and waste exchange between the fetus and its mother. The trophoblast can be modeled in vitro using trophoblast stem cells. Trophoblast stem cells require fibroblast growth factor (FGF)4, heparin, and contact with embryonic fibroblasts, or fibroblast-conditioned medium. They grow as tight epithelial colonies, which express markers of the early trophectoderm and have been shown to contribute to all of the components of the placenta through chimera studies. These cells can be passaged indefinitely and can be differentiated by removal of FGF4 and fibroblasts and will express genetic markers of later placental cell types. This chapter will discuss the initial derivation of trophoblast stem cells from the blastocyst stage, maintenance, differentiation, flow cytometry and transfection techniques that can be used with these cells.