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Protesters have been blocking the gates of the Neve Dekalim settlement, vowing fierce but non-violent resistance.
About 9,000 people have been given two days to leave all settlements in the Gaza Strip and some in the West Bank.
More than 40,000 Israeli soldiers and police are being sent to hand out eviction notices over the next two days.
Israel's Prime Minister, Ariel Sharon, said the Gaza pullout was a painful step, but essential to secure Israel's future.
In a TV address, Mr Sharon insisted Israel could not hold on to the Gaza Strip forever and pledged full support for those settlers forced to leave.
He said Palestinians would now have to prove that they were committed to peace, and that if they did, Israel would offer an olive branch in return.
Palestinian leader Mahmoud Abbas has hailed the Israeli withdrawal from Gaza as a historic moment.
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