The AU and east African bloc, the Inter-Governmental Authority on Development (IGAD), launched the investigative panel in the Ethiopian capital Addis Ababa.
The three senior military officers begin their six-week mission ahead of an August 7 deadline from Khartoum to shut a pipeline carrying South Sudanese oil for export.
In a surprise move in early June, Sudan gave companies 60 days to stop transporting oil from South Sudan after President Omar al-Bashir accused the Juba government of backing rebels in the north.
Also today, regional nations began determining the centreline of a demilitarised buffer zone that is to straddle the 2,000-kilometre undemarcated border between the two countries.
"The launch of these mechanisms underscores the seriousness with which the African Union and IGAD regard relations between Sudan and South Sudan," the AU said.
"Since 2010, Africa has been working tirelessly to promote two mutually viable states, and these current allegations threaten this objective, and in fact pose a threat to regional peace and security."
South Sudan became independent two years ago under a peace deal which ended a 22-year civil war. It took with it with most of the formerly united Sudan's 470,000 barrels per day of oil production.
The Juba government halted its crude production early last year in a dispute over how much it should pay Khartoum to use the export infrastructure.
Rising tensions led to months of intermittent border clashes, until the two countries in March reached detailed timetables to implement a deal for the oil fees and several other issues, including the demilitarised zone designed to cut cross-border rebel support.
Although the buffer zone was being set up, analysts say Bashir ordered the pipeline shut after continued rebel attacks humiliated the Sudanese authorities.
"Everybody knows that both of them are supporting rebels," an African diplomat told AFP last week. "It's clear as daylight.
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