The Prague-based "People in Need" organisation said a group of five of their staff came under fire in the northern city of Aleppo on Tuesday.
Three staff members died in the attack, while two others suffered minor injuries, the group said in a statement yesterday.
"We have all been shaken by the death of our colleagues and friends and it is with grief and pain that we are extending our condolences to the victims' families and relatives," said the group's director Simon Panek.
Despite the loss, the group vowed to continue its aid deliveries "since it is needed more and more in this deepening conflict".
The EU's aid commissioner Kristalina Georgieva said she was "deeply shocked and saddened by this dreadful news".
The civil war in Syria has claimed more than 130,000 lives and displaced millions of people since March 2011.
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