This is the first time that pro-Syrian regime forces -- which the US says includes Iranian-backed Shia militias -- have fired on the US-led coalition.
A US official told CNN that a US F-15 jet shot down the Iranian-made Shahed 129 drone, which was being controlled from a location in Syria north of At Tanf.
It was shot down after it dropped one of several weapons it was carrying near a position where coalition personnel are training and advising partner ground forces in the fight against ISIS.
The "munition did not have an effect on coalition forces," according to spokesman Col. Ryan Dillon.
"There are still some pro-regime forces that remain inside the de-confliction zone," said Dillon adding, "We remain ready to defend ourselves against any threat."
Dillon indicated the US wanted to focus on fighting ISIS rather than these forces.
A US defense official said that, for now, this was a proportional response. The munition dropped by the drone was a "dud" and is being perceived right now as a "show of force" by those who fired it.
The incident marks an escalation in the area around Al-Tanf -- a base on the Syrian-Jordanian border manned by US and British Special Forces that are advising an anti-ISIS Syrian rebel group known as Maghawir al-Thawra, or the Commandos of the Revolution.
The US-led anti-ISIS coalition also conducted another airstrike Thursday against pro-regime forces in the de-confliction zone around the coalition base, two US officials told CNN.
The strike took place after three technical vehicles were observed crossing into the restricted area. Two of the vehicles were hit about 24 miles from the At Tanf base.
This was the third coalition strike in recent weeks against pro-regime forces near At Tanf and it comes days after a highly unusual move from the coalition where they permitted a pro-regime Syrian aircraft to conduct an airstrike inside a restricted zone in southern Syria where American forces were stationed.
Tuesday's strike, which also took place near the US-manned At-Tanf Garrison on Syria's southern border, was intended to protect Iranian-backed Shia militia fighters aligned with Syrian President Bashar al-Assad who had come under attack from anti-government forces.
The US does not support the militia group but approved the strike after a request from the Syrian government was relayed by the Russians through established communication channels.
But just hours later, the US-led coalition conducted its own airstrike against the pro-regime forces after they refused to leave the de-confliction zone.
Two US defense officials outlined the course of the events to CNN providing new details into what led to the coalition's rare targeting of pro-regime forces.
Tuesday's incident unfolded in several phases over a period of about six hours and began when a small number of Iranian-backed Shia militia fighters, located just inside the US restricted zone, came under ground attack from anti-government forces.
The militia fighters were just inside a 34-mile so-called de-confliction zone the US had set up weeks earlier.
The US had been trying to keep the zone clear of outsiders in order to protect American forces but a small number of militia fighters had remained in place until they were attacked on Tuesday.
When that attack happened, the US and Russians spoke on their established de-confliction communications channel. The Russians relayed a request from the Syrian government to conduct an airstrike to support their personnel on the ground and the US agreed, according to the US officials.
Some hours later, the US and Russia spoke again -- this time the Russians passed along a request to evacuate wounded personnel from the restricted area using unarmed vehicles.
It wasn't immediately clear how many wounded were evacuated.
But a short time later, the pro-regime forces begin bringing additional fighters and equipment into the area and when they refused to leave, the US carried out its own airstrike targeting the militia forces.
"Despite previous warnings, pro-regime forces entered the agreed-upon de-confliction zone with a tank, artillery, anti-aircraft weapons, armed technical vehicles and more than 60 soldiers posing a threat to coalition and partner forces based at the At Tanf Garrison," a coalition statement said on Tuesday.
After several warnings over the de-confliction channel, the coalition destroyed "two artillery pieces, an anti-aircraft weapon, and damaged a tank."
The airstrike was carried out by a US F/A-18 aircraft that dropped four bombs in total and killed an estimated 10 fighters in addition to the previously reported damaged and destroyed vehicles.
Last week, the coalition said that a relatively "small element" of pro-regime forces continued to hold their position inside the "de-confliction" zone surrounding the coalition training base.
Coalition spokesperson, Col. Ryan Dillon, told reporters last week that the pro-regime forces inside the zone represented an "armed and hostile presence" and "they pose a threat to us in that particular location."