Molecular virological understanding of the feline immunodeficiency virus (FIV) life cycle is increasing, facilitating rational derivation of improved vectors from this non-primate lentivirus. The packaging signal has been mapped, a central DNA flap has been identified, and class I integrase mutants have been validated. Vector systems with improved effectiveness and safety profiles are being applied by a number of laboratories in several pre-clinical models, with demonstrated efficacy in human tissues. The comparative lentivirological research that facilitates FIV vector development may also yield insights into the still enigmatic molecular basis for a signature lentiviral property with pathogenetic and therapeutic importance: permanent transgene integration in non-dividing cells. This review discusses virological aspects of lentiviral vector development, as well as some recent controversies and applications.