Little Apollo Gabriel Rodrigues caught the minibus around 7am on Tuesday (14 Nov) to get to nursery school.
But the driver and his assistant allegedly failed to drop him off with the other kids and left him in the vehicle in the car park.
He was later found unresponsive in the penultimate row of seats.
The toddler was rushed to hospital around 4.20pm, but medics could only pronounce him dead.
Devastated mum Kaliane Rodrigues told the media in São Paulo, Brazil: “He was doing so well.
“But when I went to put him in the minibus, he cried. He cried. Didn’t want to go.”
Police arrested and questioned driver Flávio Robson Benes, 45 and his assistant, 44-year-old Luciana Coelho Graft.
Graft, who is also Benes’s wife, was said to have told officers she was having a migraine at the time, which may have affected her attentiveness.
They were released on bail after a custody hearing on Wednesday (15 Nov).
The autopsy report is still pending, but police believe Apollo died from heat-related causes.
Highs of 37.7 C (99.8F) were recorded in São Paulo that day.
Police are investigating the case as suspected child homicide.
The local authorities have appointed a team of psychologists to provide counselling to Apollo’s family.
Kaliane told local media: “I just want justice.”