CAR T cell therapy has shown remarkable efficacy in treating hematological tumors, yet challenges remain in efficiently producing potent CAR T cells due to complex protocols. Our study provides important insights into spinoculation methods for gene delivery and viral vector production, which could optimize CAR T cell production.Our data indicate that gene transduction is signifcantly dependent on speed and duration of centrifugation, concentration and number of viral particles, the concentration of polybrene, and number of infected T cells. In addition, we optimized DNA ratios transfected into 293T cells to produce high titer of viral particles.