Feline immunodeficiency virus (FIV)-based lentiviral vectors are useful for introducing integrated transgenes into nondividing human cells. This protocol describes the production of FIV-based lentiviral vectors using cells grown in CF10 or CF2 devices. It also details the harvesting and concentration of these vectors. The methods are for production for basic science laboratory use and in vivo experimentation. They do not meet standards for clinical-grade (good manufacturing practice [GMP]) production.