Lentiviral transduction is a gene delivery method that provides numerous advantages over direct transfection and traditional retroviral or adenoviral delivery methods. It facilitates for the transduction of primary cells inherently difficult to transfect, delivers constructs of interest to nondividing as well as dividing cells, and permits the long-term expression of sizable DNA inserts (e.g., <7 kb). The study of peripheral nerve myelination at the molecular level has long benefited from the Schwann cells/dorsal root ganglia (DRG) neurons myelinating co-culture system. As this culture system takes about a month to develop and perform experiments with, lentiviral-delivered constructs can be used to manipulate gene expression in Schwann cells and DRG neurons, primary cells that are otherwise resilient to direct transfection. Here we present our protocol for lentiviral production and purification and subsequent infection of large numbers of Schwann cells and/or DRG neurons for the molecular study of peripheral nerve myelination in vitro.