Crossrail is the UK's biggest transport project since the Channel Tunnel
Heathrow Airport operator BAA has announced a £230m funding package for the £16bn Crossrail project.
The scheme will connect Maidenhead in Berkshire to Shenfield in Essex via Heathrow and central London.
The project received the Parliamentary go-ahead in July with the first trains expected to start running from 2017.
The rail project is the UK's biggest transport project since the Channel Tunnel and is expected to create up to 30,000 jobs, experts believe.
The new rail route will provide four fast trains an hour to Heathrow and will also run direct services from Heathrow airport through central London, the City and Canary Wharf.
New stations will be created at Paddington, Bond Street, Tottenham Court Road, Farringdon, Liverpool Street, Whitechapel and the Isle of Dogs (Canary Wharf).
Transport minister Lord Adonis and BAA's strategy director Mike Forster agreed on the deal.
Lord Adonis said: "Crossrail will definitely go ahead and the announcement of £230m from BAA further underpins the credibility of the project and ensures that the work will proceed from 2010 so that it could be open in 2017."
The government and the mayor of London are providing the bulk of the project's cost while Lord Adonis has assured observers that despite the economic downturn businesses in London would keep their funding promise.
The City had agreed to make a "significant contribution to the future of Crossrail", he said.
Lord Adonis added: "Crossrail will be transformational for transport in London creating the first comprehensive east-west connections that we have had in a generation in terms of new links."
Mr Forster said: "We are delighted to support the Crossrail scheme, which demonstrates our commitment to improving public transport access to Heathrow."
The Confederation of British Industry's deputy director-general John Cridland said: "As well as giving the construction industry a significant boost at a difficult time, Crossrail will play a vital role in improving London's transport network and enhancing its reputation as a world-class city to do business in.
"With this major plank of funding in place from BAA, it is important that there are no further delays to Crossrail and that work begins as soon as possible."
The 74-mile route (118.5km) will be underground for 26 miles (41.5km) and allow for stops at 38 stations.