A defective interfering (DI) influenza virus carries a large deletion in a viral gene segment and shows potential for antiviral therapy by interfering with the replication of infectious virus. However, because a DI virus cannot replicate autonomously without the aid of an infectious helper virus, clonal DI virus stocks without contamination of the helper virus have not been generated. To overcome this problem, we generated a clonal DI virus with a PB2 DI gene by reverse genetics, amplified the clonal DI virus using a cell line stably expressing the PB2 protein, and confirmed its ability to interfere with infectious virus replication in vitro. Thus, our approach is suitable for obtaining purely clonal DI viruses, will contribute to the understanding of DI virus interference mechanisms, and can be used for the development of DI virus-based antivirals.