A message from Valerie Amos, seen on Thursday, says the expulsions this month "set a worrying precedent for all humanitarian staff in Syria, in that any staff member in contact with armed opposition groups for the purpose of negotiating access is potentially under threat of being declared persona non grata."
The United Nations and other aid groups have struggled to get badly needed aid to millions of people in Syria's civil war, which moves into its fifth year next month.
Syria's government has been blamed repeatedly for hindering the effort, which is also complicated by multiple armed groups and the recent rise of the Islamic State militant organisation.
Amos' message to colleagues indicates that the effort to secure a six-week suspension of government airstrikes and artillery shelling in Aleppo is at risk.
The UN special envoy for Syria, Staffan de Mistura, was heading to Damascus on Saturday to work out details on the proposed freeze in hostilities throughout the devastated city, which would allow the UN to test the delivery of unrestricted humanitarian aid to one district there.
"Whilst the Government of Syria is giving public support to the Special Envoy's freeze initiative, they are making it difficult to actually implement it, should agreement be reached by the Special Envoy with all parties," Amos wrote.
"The Government has removed a key humanitarian staff member with the contacts to negotiate access with a wide range of armed opposition groups in Aleppo, and who was expected to play a leading role in the event that the freeze materialises."
The expulsions also will have a significant impact on the entire humanitarian operation for Syria, as the two staffers played a critical role in helping to coordinate inter-agency
aid convoys and maintained direct contact with key armed opposition groups for negotiating aid access, Amos wrote.
She said one of the two expelled staffers had extensive contacts with such groups in Aleppo.
Syria's foreign affairs ministry on February 9 declared one of the international staffers, who was working in Homs governorate, persona non grata and had one week to leave the country, Amos wrote. That staffer has since left.