Papers setting out the EU's Brexit talks strategy say that the region will only be included in a post-Brexit trade deal if Spain agrees with the terms.
Gibraltar's chief minister Fabian Picardo has warned against Gibraltar being used as a bargaining chip during Brexit negotiations.
Some 96% of Gibraltar's citizens voted to remain in the EU.
Emily Thornberry the shadow foreign secretary said: "The issue of Gibraltar is clearly a hard and thorny issue, but the truth is the people of Gibraltar want to remain British and they should do".
"Brexit is already complicated enough without Spain trying to complicate it further".
The rock has in recent years become a haven for gaming, banking and financial services companies which attract more than 10,000 cross-border workers."The U.K. Government's position is clear and will stand". Hence the European Union is likely to favor Spanish demands.
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Tusk's document said: "In these negotiations the Union will act as one".
Esteban González Pons, the vice-chairman of the European People's Party, told Spain's El Pais newspaper this omission was "very relevant", adding: "Gibraltar isn't part of the United Kingdom". Gibraltar has remained on the list ever since.
In 2002 a referendum which would have given Spain and Britain shared control of the region was rejected by nearly 99% of the population.
Spain has a long-standing territorial claim on British Overseas Territory Gibraltar, held by the United Kingdom since 1713, which now houses key British army bases.
Mr Picardo added: "What we are seeing is a clear manifestation of the predictably predatory attitude that we anticipated Spain would seek to abusively impose on its partners, as they have been threatening to do since the referendum took place and as the only member of the European Union with an obsession with Gibraltar".
The clause sparked an outcry among U.K. Conservative MPs.
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Theresa May did not refer directly to Gibraltar in her Article 50 letter to Donald Tusk on Wednesday.
"We are clear that Gibraltar is covered by our exit negotiations".
"We have been firm in our commitment never to enter arrangements under which the people of Gibraltar would pass under the sovereignty of another state against their wishes, nor to enter into a process of sovereignty negotiations with which Gibraltar is not content", May said.
"It's predictable that, given Spain's previous behavior, they would try and use Brexit as a fig leaf for trouble-making over the status of Gibraltar", Lopresti said Friday.
A clause has been inserted in the EU's draft Brexit negotiating guidelines that appears to allow Spain to exclude Gibraltar from any transitional single market access arrangement or future trade deal with the United Kingdom if it is not satisfied with the status of the territory.
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