The present invention relates to a method for obtaining an untransformed avian cell line enabling in vitro avian virus replication. Said method includes the following steps: a) culturing avian embryonic stem cells in the presence of a stroma for at least 3 days b) culturing for at least 2 days in a medium having a low serum concentration c) culturing for at least 2 days in a medium having a low serum concentration containing 1 to 10 mM of hexamethyleme bisacetamide (HMBA) d) culturing for at least 10 days in a medium having a low serum concentration and e) culturing or freezing an avian cell line enabling avian virus replication. The invention also relates to the resulting cell line and to the use thereof in vaccine preparations.
