Lentiviral vectors efficiently transfer genes into a broad spectrum of cells and tissues, including non-dividing cells and stem cells. Lentiviruses integrate their viral genome into the host chromosome, which is the basis for virus latency as well as stable transgene expression. A rather novel development is the use of lentivectors to transfer transgenes in oocytes and early embryos to generate transgenic animals, a technology also known as lentiviral transgenesis. Lentiviral transgenesis has been shown to be highly efficient in many different species, including mouse, rat, pig, bovine, and even birds. Thus, lentiviral transgenesis has the potential to become a versatile and widespread transgenic technology. The aim of this chapter is to cover important practical aspects of lentiviral transgenesis, including vector preparation, gene delivery into the early embryos and lentiviral RNA interference.