Some say President Putin plays EU members against each other
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The European Union has warned Russia that it must extend an existing trade and political agreement to the 10 countries set to join the bloc in May.
Any failure to do so will have serious repercussions, officials say.
Foreign ministers who met in Brussels on Monday told Moscow that any concerns it may have about EU expansion should be discussed separately.
The EU is reassessing the troubled relationship with its biggest neighbour as it prepares to spread eastwards.
This spring, both the EU and Nato will expand deep into the former Soviet bloc and Russia is watching this double expansion with concern.
The BBC's Oana Lungescu in Brussels says there is not much it can do about Nato, but it is refusing to extend an existing agreement with the EU to the new member countries unless it gains more trade concessions and visa-free travel for its citizens.
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Moscow is also seeking guarantees on the status of ethnic Russians in the former Soviet republics set to join the EU.
But EU foreign ministers warned Russia that the agreement has to apply to all countries of the bloc by 1 May to avoid what they call a "serious impact" on EU-Russia relations.
An EU official said that otherwise Russia would be in breach of the agreement and possible sanctions would have to be considered.
As the list of open rows with Russia grows longer, the EU is trying harder than before to speak with one voice rather than allow President Vladimir Putin to play off bigger EU countries against the rest, Oana Lungescu says.