The 300th Anniversary of Mayflower's Landing was commemorated in 1920 and early 1921 by celebrations throughout the United States and by countries in Europe. Delegations from England, Holland and Canada met in New York. The mayor of New York, John Francis Hylan, in his speech, said that the principles of the Pilgrim's Mayflower Compact were precursors to the United States Declaration of Independence. While American historian George Bancroft called it "the birth of constitutional liberty."Governor Calvin Coolidge similarly credited the forming of the Compact as an event of the greatest importance in American history.

However, life in Holland became increasingly difficult for the congregation. They were forced into menial and backbreaking jobs, such as cleaning wool, which led to a variety of health afflictions. In addition, a number of the country's leading theologians began engaging in open debates which led to civil unrest, instilling the fear that Spain might again place Holland's population under siege, as it had done years earlier. England's James I subsequently formed an alliance with Holland against Spain, with a condition outlawing independent English church congregations in Holland. Thus formed the separatists' motivating factors to sail for the New World, with the benefit of being beyond the reach of King James and his bishops.

Their desire to travel to America was considered audacious and risky, as previous attempts to settle in North America had failed. Jamestown, founded in 1607, saw most of its settlers die within the first year. 440 of the 500 new arrivals died of starvation during the first six months of winter. The Puritan separatists also learned of the constant threat of attacks by Indigenous peoples. But despite all the arguments against traveling to this new land, their conviction that God wanted them to go held sway: "We verily believe and trust the Lord is with us," they wrote, "and that He will graciously prosper our endeavors according to the simplicity of our hearts therein."

During mid-July of 1620, sixty-five passengers aboard the Mayflower left from where it was docked in Rotherhithe. The Mayflower made its way from the Thames River and into the English Channel. From there, it reached Southampton Water and anchored there for a week. After seven days, the Mayflower joined a group of Leiden church members aboard the Speedwell that had come from Holland. Two weeks later, the Mayflower and the Speedwell set sail on about August 5, 1620. The Speedwell quickly had to go into repairs in Dartmouth after it sprang a leak. When repairs were done, the ships set sail again, but the Speedwell sprang yet another leak not long after, It was early September by this point, and the Speedwell was abandoned. Eleven of its passengers boarded the Mayflower, while the rest stayed in London.

In explaining to his congregation why they should emigrate, Robinson used the analogy of the ancient Israelites leaving Babylon to escape bondage by returning to Jerusalem, where they would build their temple. "The Pilgrims and Puritans actually referred to themselves as God's New Israel," writes Peter Marshall. It was therefore the manifest destiny of the Puritans to similarly build a "spiritual Jerusalem" in America.

When it was time to leave, the ship's senior leader, Edward Winslow, described the scene of families being separated at the departure: "A flood of tears was poured out. Those not sailing accompanied us to the ship, but were not able to speak to one another for the abundance of sorrow before parting." William Bradford, another leader who would be the second Governor of the Plymouth Colony, similarly described the departure:

Truly doleful was the sight of that sad and mournful parting. To see what sighs and sobs and prayers did sound among them; what tears did gush from every eye, and pithy speeches pierced each heart...their Reverend Pastor, falling down on his knees, and they all with him...