Human challenge studies are important tools in influenza research, especially during intensified efforts to develop a universal influenza vaccine. However, such studies of influenza viruses are often hampered by the lack of access to relevant, good manufacturing practices-grade challenge viruses. This issue is further complicated by different influenza viruses preferring different growth substrates; thus, one production process does not work for all viruses. Here, we sought to produce a recombinant master virus seed suitable for development as a human challenge virus by using viral production cells in suspension. Our initial attempts at utilizing adherent Vero cells resulted in suboptimal virus rescue and propagation properties, and virus rescue efficiency, using plasmid-based reverse genetics, was extremely poor as compared to that seen when adherent 293T cells were used. HEK-293 cells are extensively used as viral production suspension cells for Lentivirus and Adeno-associated virus production. Using quality-assured viral production cells (HEK-293) in suspension, we optimized the process for rescue and propagation of the A/Texas/71/2017 (H3N2) influenza virus and produced a master virus seed under good manufacturing practice conditions. This work will facilitate human challenge studies, which may help accelerate the development of a universal influenza vaccine.