Plasmid-based reverse genetics systems allow the generation of influenza A virus entirely from cloned cDNA. However, since the efficiency of virus generation is dependent on the plasmid transfection efficiency of cells, virus generation is difficult in cells approved for vaccine production that have low transfection efficiencies (e.g., Vero cells). Here we established an alternative reverse genetics system for influenza virus generation by using an adenoviral vector (AdV), which achieves highly efficient gene transfer independent of cell transfection efficiency. This AdV-mediated reverse genetics system will be useful for generating vaccine seed strains and for basic influenza virus studies.