Lentiviral vectors have been successfully used in the clinic and they are increasingly being used for nonclinical applications. They are capable of stably transducing a broad range of mammalian cell types, including nondividing cells, with high efficiency. This review summarizes the evolving molecular design of lentiviral vectors, describing how they have improved since their first description. Lentiviral vector safety and issues surrounding genotoxicity are discussed. Examples of successful application of lentiviral vectors in laboratory and preclinical research are described. These include functional genomics, target validation, protein manufacturing, in vivo imaging, transgenic animals, and stem cell research.