A treaty conference was held in 1744 between representatives of the colonial governments of Pennsylvania, Maryland, and Virginia and delegates from the Onondaga, Oneida, Cayuga, Seneca, and Tuscarora tribes of the Iroquois Confederation. The purpose was to settle disputes with the Iroquois over the ownership of lands at the western margin of the three colonies. The representatives from Virginia at the conference were Thomas Lee and William Beverley, with William Black as secretary.

At the conclusion of the conference, the Virginia commissioners believed they held a deed for all of the land west of Virginia to the Mississippi River and north to the Great Lakes. This included all of present West Virginia. However, the Iroquois believed that they had sold land only in Virginia south of the Potomac River and between the Blue Ridge Mountains and the Allegheny Mountains, excluding most of the present state of West Virginia. The disagreement over the land title continued until the Iroquois formally relinquished all ownership rights to the disputed territory during the Treaty of Fort Stanwix in 1768.

The Treaty of Lancaster was a milestone in the history of Western Virginia. It allowed more than 1,000 European immigrants to settle in what is now eastern West Virginia between 1745 and 1755 unopposed by the Iroquois and other tribes. These pioneers established a firm enough toehold that not all were driven away during the French and Indian War (1754—63). Those that remained after the war set the stage for the remainder of Western Virginia to be settled at a rapid pace during the last quarter of the 18th century.