Representative Nancy Pelosi, former House speaker and a key ally of Joe Biden, signed a letter on Friday from dozens of congressional Democrats to the president and Secretary of State Antony Blinken, urging a halt to weapons transfers to Israel.
More than 33,000 people have died in Israel's war on the narrow coastal enclave, where a famine is also imminent and diseases are widespread, UN agencies have warned.
Support from Pelosi, a veteran member of Biden's Democratic Party, for stopping the transfer of weapons to Israel demonstrates that the view is increasingly becoming mainstream in the party.
Friday's letter called on the Biden administration to conduct its own probe into an Israeli airstrike that killed seven staff of the aid group World Central Kitchen on Monday.
"In light of the recent strike against aid workers and the ever-worsening humanitarian crisis, we believe it is unjustifiable to approve these weapons transfers," the letter said. It was signed by Pelosi and 36 other Democrats including Representatives Barbara Lee, Rashida Tlaib and Alexandria Ocasio-Cortez.
This comes despite the former House Speaker coming under fire in January for suggesting that some pro-Palestine protesters were linked to Russia, and urged the FBI to investigate.
Her comments were dismissed as "unsubstantiated smears" by the Council on American-Islamic Relations (CAIR).
Pro-Palestinian protesters responded to Pelosi by delivering 300lbs of manure to be dumped outside her front yard.
The Israeli military said on Friday it dismissed two officers and formally reprimanded senior commanders after an inquiry into the aid workers' deaths found serious errors and breaches of procedure - an incident that drew worldwide condemnation.
Biden held a call with Israeli Prime Minister Benjamin Netanyahu on Thursday in which he said Israel needed to do more to protect civilians or the US would change its policy.