Lentiviral vectors have become a promising new tool for the establishment of transgenic animals and the manipulation of the mammalian genome. While conventional microinjection-based methods for transgenesis have been successful in generating small and large transgenic animals, their relatively low transgenic efficiency has opened the door for alternative approaches, including lentiviral vectors. Lentiviral vectors are an appealing tool for transgenesis in part because of their ability to incorporate into genomic DNA with high efficiency, especially in cells that are not actively dividing. Lentiviral vector-mediated transgene expression can also be maintained for long periods of time. Recent studies have documented high efficiencies for lentiviral transgenesis, even in animal species and strains, such as NOD/scid and C57Bl/6 mouse, that are very difficult to manipulate using the standard transgenic techniques. These advantages of the lentiviral vector system have broadened its use as a gene therapy vector to additional applications that include transgenesis and knockdown functional genetics. This review will address the components of the lentiviral vector system and recent successes in lentiviral transgenesis using both male- and female-derived pluripotent cells. The advantages and disadvantages of lentiviral transgenesis vs. other approaches to produce transgenic animals will be compared with regard to efficiency, the ability to promote persistent transgene expression, and the time necessary to generate a sufficient number of animals for phenotyping.