Southampton were kicked out of the play-offs after admitting spying on three clubs during the Championship season.
Middlesbrough, beaten by Southampton in the semi-finals, have been reinstated and will now play Hull City for a place in the Premier League.
The EFL commissioned the Saints to watch Oxford United and Ipswich Town training, as well as filming Middlesbrough as they prepared for the first leg of their play-off semi-final on 7 May.
The independent disciplinary commission also awarded Southampton a four-point deduction in the Championship for next season.
The Saints will now miss out on a match considered the richest in world football, with the winners of the play-off final guaranteed a minimum of £110m in Premier League broadcast revenue.
The final will take place on Saturday at Wembley and the start time is to be confirmed.
Southampton admitted “multiple breaches of EFL regulations relating to unauthorized filming of other clubs’ training sessions”, the EFL said.
The club has also received a reprimand for all charges.
Sources have confirmed to BBC Sport that Southampton will lodge an appeal on Wednesday and argue that the punishment is disproportionate.
The EFL said it would be “working to try to resolve any appeals on Wednesday 20 May”.
He added that “subject to the result, it could result in a further change to Saturday’s match.”
The appeal will be heard by an Independent League Arbitration panel with three new members.
The EFL said in a statement: “Southampton was first charged on Friday 8 May, with further charges issued on Sunday 17 May in relation to additional breaches during the 2025-26 season.
“Those additional charges arose from matters identified after the initial proceedings involving Middlesbrough were commenced.
“Southampton admitted breaches of regulations which require clubs to act with the utmost good faith and prohibit observing another club’s training session within 72 hours of a scheduled match.
“The admitted infringements relate to matches against Oxford United in December 2025, Ipswich Town in April 2026 and Middlesbrough in May 2026.”
Southampton did not win any of the three games: they lost 2-1 at Oxford, drew 2-2 at home against Ipswich and 0-0 at Boro.
The statement continued: “The EFL is now in discussions with all three clubs about the implications of today’s decision and will make a further announcement in due course.”
Middlesbrough issued a statement saying they “welcomed the result”.
“We believe this sends a clear message for the future of our game regarding sporting integrity and conduct,” he added.
“As a club we are now focused on our match against Hull City at Wembley on Saturday. Ticket information for our supporters will be available shortly.”
Southampton had already sold out tickets for Saturday’s match and fans will receive a full refund.
There has yet to be a response from Southampton, but a source sympathetic to the situation said the club had not been given the precise reasons for the decision and that their lawyers expected to have to work all night on it.
Southampton’s chief executive sent an email to staff saying the club is confident it can reverse the decision.
It is understood that all parties have agreed to take the matter to a conclusion on Wednesday to avoid the possibility of a final decision being postponed.
However, they must also wait to see whether the Football Association brings charges against those involved in the espionage. The EFL can only apply sanctions against its member clubs.
After a spying case at the 2024 Olympics, FIFA banned three members of Canada’s staff, including the head coach, from participating in all football for a year.