The success of experimental gene therapy is dependent on the ability to safely and efficiently introduce transgenes into the target cell or tissue. Retroviral-based vectors, notably those derived from Moloney murine leukemia virus (MLV) and lentiviral vectors derived from HIV, have proven to be valuable gene transfer vehicles as a result of their ease of production and their ability to mediate long-term transgene expression. One of the most widely used methods for viral vector production is based on the transient transfection of viral vector plasmid DNA into a producer cell line. Here, we describe protocols to produce and standardize high quality MLV-based retroviral and HIV-based lentiviral vectors for ex vivo and in vivo gene delivery.