Trivalent live cold–adapted attenuated reassortant influenza vaccines (LAIVs) have been successfully used for years in Russia (Ultravac) and in the USA (FluMist) to control influenza. The reassortant vaccine virus comprises hemagglutinin (HA) and neuraminidase (NA) genes from the circulating viruses and the remaining six genes from the cold–adapted master donor virus (MDV). The LAIV phenotypic properties cold adaptation (ca), temperature sensitivity (ts) and attenuation (att) are inherited from the six genes encoding the internal proteins of the MDV. The main focus of this chapter is to explore the role of the gene segments of MDV as it relates to the attenuated phenotype.