The Centre will be staffed by experts from Estonia, Germany, Italy, Latvia, Lithuania, Slovakia and Spain.
"The need for a cyber-defence centre is compelling," said General James Mattis, Nato's Supreme Allied Commander Transformation, at the signing ceremony. "It will help Nato defy and successfully counter the threats in this area."
The attacks on Estonia sparked interest in online warfare when the country, which has pioneered electronic government, came under sustained attack from computers around the world.
The attack is thought to have been the work of a flash mob of predominantly Russian computer users protesting against the removal of a memorial in Estonia.
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