Background & Aim The use of viral vectors for gene therapy applications is an emerging market that has gained considerable clinical traction in recent years. As the demand for gene therapy solutions has increased, the ability to produce viral vectors at manufacturing scale has become more critical. The Gibco™ CTS™ LV-MAX™ Lentiviral Production System is a cGMP-manufactured, xeno-free, 293F-based suspension platform that produces lentiviral vectors with high infectious titers (>1 x 108 TU/mL). The CTS LV-MAX system comprises GMP-banked 293F Virus Production Cells (VPCs), chemically defined growth and production medium, as well as a high-efficiency transfection reagent, titer boosting supplement and novel production enhancer. Since VPCs lack the large T antigen and have been adapted for high density growth in serum-free suspension culture, the LV-MAX system is inherently more scalable and cost-effective than traditional adherent-based virus production platforms. Here, we describe the scale up of the CTS LV-MAX system into stirred tank bioreactors and provide guidance for large scale production of lentiviral vectors under serum-free, suspension conditions. Methods, Results & Conclusion Design of experiment (DOE) and statistical analyses were used to identify the impact of various factors impacting lentiviral production in the CTS LV-MAX System including pH, stir speed, and timing of various reagent additions in both the ambr® 15 microbioreactor system and 3L Thermo Scientific™ HyPerforma™ stirred tank bioreactors. This multi-platform experimental approach allowed for the rapid development of a robust 2L working volume stirred tank bioreactor process capable of generating lentiviral vectors at levels equivalent to those of traditional small scale shake flasks. Future efforts to scale up production into larger bioreactors will further serve to meet the overwhelming production needs currently facing the gene therapy industry.