Human influenza vaccine reference strains are prepared as required when an antigenically new strain is recommended by WHO for inclusion in the vaccine. Currently, for influenza A, these strains are produced by a double infection of embryonated hens' eggs using the recommended strain and the laboratory strain PR8 which grows to high titre in eggs, in order to produce a high growth reassortant (HGR). HGRs are provided by WHO reference laboratories to the vaccine manufacturing industry which use them to prepare seed virus for vaccine production. The use of reverse genetics in preparing vaccine reference strains offers several advantages over the traditional method: (i) the reverse genetics approach is a direct rational approach compared with the potentially hit-or-miss traditional approach; (ii) reverse genetics will decontaminate a wild type virus that may have been derived in a non-validated system, e.g. a cell line not validated for vaccine purposes, or that may contain additional pathogens; (iii) at the plasmid stage, the HA can be engineered to remove pathogenic traits. The use of reverse genetics in deriving HGRs has been demonstrated by several laboratories, including its use in deriving a non-pathogenic reassortant strain from a highly pathogenic virus. In this report, we have advanced the use of reverse genetics by making use of a cell line acceptable for human vaccine production, by demonstrating directly the short time frame in which a reassortant virus can be derived, and by deriving a non-pathogenic pandemic vaccine reference virus in cells validated for vaccine production and under quality controlled conditions.