IMPORTANCE The live attenuated influenza vaccine FluMist® has been proven safe and effective and are widely used in the USA. The phenotype and genotype of the vaccine virus are believed to be very stable and mutants that cause disease in animals or humans have never been reported. By propagating the virus under well-controlled laboratory conditions, we found that the FluMist vaccine backbone could regain virulence to cause severe disease in mice. The identification of the responsible substitutions and elucidation of the underlying mechanisms provide unique insights on the attenuation of influenza virus, which is important to basic research on vaccines, attenuation reversion, and replication. In addition, this study suggests that the safety of LAIVs should be closely monitored after mass vaccination and novel strategies to continue to improve LAIV vaccine safety should be investigated.