To investigate the molecular changes that allow influenza B viruses to adapt to new mammalian hosts, influenza B/Florida/04/2006 was serially passaged in BALB/c mice until highly virulent. The viral factors underlying this transition were then investigated in mice and ferrets. Five viruses, including the wild-type virus (P0), three intermediate viruses (P5, P9, and P12), and a lethal mouse-adapted virus (P17 (MA)), harbored one to five amino acid substitutions in the hemagglutinin, M, NP, and PA segments suggesting that these mutations enhance virulence. The P17 (MA) virus replicated significantly more efficiently than the P0 virus both in vitro and in vivo (P < 0.0001), and was highly virulent (MLD50: 10(5.25)TCID50) while the P0, P5, and P9 viruses did not kill any infected mice (MLD50 > 10(6.0)TCID50). Furthermore, the P17 (MA) virus grew to greater titers in the ferret upper respiratory tract compared with the P0 and intermediate viruses, and only the P17 (MA) virus was transmissible between ferrets via both direct and aerosol contact. To our knowledge, this is the first study to demonstrate ferret-to-ferret transmission of influenza B virus and to delineate factors that may affect its transmission.