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The UK king, who is president of the British Red Cross, discussed the “acute humanitarian situation” in the region, a Buckingham Palace representative said.
"We are immensely grateful to HM King Charles for his generous donation to our Israel and Occupied Palestinian Territory appeal," the charity said on social media.
The monarch also met charity leaders from Medical Aid for Palestinians, Unicef UK and Christian Aid.
Aid groups have called for a ceasefire in the Israel-Gaza conflict to protect civilians and humanitarian workers, and ensure safe and sustained access to aid.
UK Prime Minister Rishi Sunak has emphasised the need for temporary breaks in the bombings to allow Israeli hostages and British citizens to be freed.
Buckingham Palace did not comment on whether the king discussed the hostages during the private meeting.
The International Committee of the Red Cross, in its neutral intermediary role, has assisted with the recent release of four Israeli hostages. Hamas is still holding more than 200 people.
The latest from the Israel-Gaza war - in pictures
Those the audience were the chief executive of Unicef UK Jon Sparkes, Medical Aid for Palestinians chief executive Melanie Ward, vice chairwoman of the board of trustees for Medical Aid for Palestinians Shireen Jayyusi, Christian Aid chief executive Patrick Watt, and Rory Moylan, head of region for the Middle East, North Africa and Europe at the British Red Cross.
The British Red Cross has described the situation in Gaza as an “unprecedented humanitarian crisis” with vital resources unable to get through.
It said the most pressing issue is the lack of food and water and that recent aid convoys were merely “a drop in the ocean”.
Israel has only in recent days agreed to allow aid into Gaza, having besieged the Hamas-ruled area, preventing essentials such as water, food and fuel from reaching more than two million Palestinians.
But the UN has warned it is on the verge of running out of fuel in the Gaza Strip, forcing it to sharply curtail relief efforts in the territory.
The king also held an audience with the Crown Prince of Kuwait on Wednesday, when he also discussed the continuing conflict, which has already claimed at least 8,000 lives on both sides.
Israeli Prime Minister Benjamin Netanyahu has said the military is preparing for a full-scale ground invasion to crush Hamas.
In a landmark speech last week, King Charles stressed the “vital” need for mutual understanding among religions in times of “international turmoil and heart-breaking loss of life”.
Earlier in the month, he held talks with Sir Ephraim Mirvis, the Chief Rabbi, at the palace to discuss the horrors of events in Israel and how to support interfaith harmony in Britain in distressing and difficult times.
Buckingham Palace said King Charles condemned Hamas’s “barbaric acts of terrorism” after its October 7 attack on Israel – which killed 1,400 people.