Deputies said the crash happened around 9:15 a.m., Saturday on soutbound Highway 27 at Polo Park Boulevard in Davenport.
Robert Carroll, 41, of Clermont, was driving a Toyota with his two children, 4-year-old Nicholas Jayden Carroll and 2-year-old MacKenzie Carroll in the back, according to a release.
The 41-year-old father was stopped at the intersection when a box truck operated by 18-year-old Ednaldo Silva Marques of Winter Garden came upon the traffic at highway speed, deputies said.
Deputies said the driver tried to stop using the emergency brakes, but could not avoid hitting the Toyota.
Deputies said the impact from the crash forced the back of the Toyota into the back seat where the children were sitting strapped into their car seats. Deputies said Nicholas Jayden Carroll was killed.
The Toyota was forced into another car. The two people inside were not hurt, deputies said.
Two-year-old MacKenzie is at Arnold Palmer hospital in serious condition, deputies said.
Their father was taken to Orlando Regional Medical Center and is listed in guarded condition, deputies said.
The box truck driver was released from South Lake Hospital with minor injuries he incurred trying to break the window of the Toyota to help the Carroll family, deputies said.
The Polk County Sheriff's Office said none of the drivers were impaired.
Southbound U.S. 27 was closed for about five hours due to the crash.
Deputies do not anticipate criminal charges.