The League Arbitration Panel's decision is final and cannot be appealed further.
"Southampton Football Club has this evening been informed that the EFL’s League Arbitration Panel has upheld the sanctions imposed earlier this week. As a result, the club will not participate in Saturday’s Championship Play-Off Final," the club said on their official site.
"This is an extremely disappointing outcome for everybody connected with Southampton Football Club. We know how painful this moment will be for our supporters, players, staff, commercial partners and the wider community who have given so much backing to the team throughout the season and we apologise once again to everyone impacted by this.
"While we fully acknowledge the seriousness of this matter and the scrutiny that has followed, the club has consistently believed the original sporting sanction was disproportionate, a view that has been widely shared by many in the football community over the last 24 hours.
"We would like to place on record our sincere thanks to our supporters for the support, patience and loyalty they have shown throughout an incredibly difficult period. We will share information as soon as possible in relation to ticket refunds for those who bought tickets to Wembley.
"Southampton Football Club has a proud history and strong foundations, but it is clear that trust now needs to be rebuilt. That work begins immediately. The club will reflect carefully on the events that have led to this point, learn from them and take the necessary steps to move forward responsibly.
"While tonight is a painful moment, this football club will respond with humility, accountability and determination to put things right."
Meanwhile, Hull are reportedly considering consulting lawyers to see if there is a case that they can be promoted directly to the Premier League, and are especially unhappy that they have been given limited time to prepare for Middlesbrough, having thought they were taking on Southampton.
The Championship play-off final will take place on Saturday, May 23rd at 16:30 CET.