The second Spanish occupation of Florida was short-lived and constantly threatened by American expansion. West Florida included parts of present-day Alabama, Mississippi, and Louisiana, along with what are now the 10 counties of western Florida. Most of this area was claimed by the United States as part of the Louisiana Purchase of 1803. (Spain had secretly ceded most of its holdings in the area to France in 1800, but later held that it still owned both West and East Florida.)

American settlers declared the western section of West Florida (up to the Pearl River) independent in 1810; it was made a part of the new state of Louisiana in 1812. The United States seized the central part of West Florida (between the Pearl and Perdido rivers) and added it to Mississippi Territory in 1813. After several Spanish towns were captured by General Andrew Jackson in the First Seminole War (1817–18), Spain reluctantly relinquished control of East Florida to the United States. It also gave up its claims to all of West Florida in a treaty ratified in 1821. Jackson was named provisional governor, with headquarters at Pensacola.

In 1822 the Territory of Florida was organized, and a civil government set up. William P. Duval was chosen the first territorial governor. In 1824 Tallahassee was selected as the site for the capital.

In 1823 a number of tribal chiefs signed the treaty of Moultrie Creek, which relinquished most Indian lands in favor of a reservation area. Later, however, the government attempted to move the Seminoles out of Florida, and some Indians, led by Osceola, resisted. The long and bloody Second Seminole War (1835–42) resulted. The majority of the Indians were subdued and forced to move to Indian Territory (now Oklahoma), but several hundred undefeated Seminoles remained in the Everglades region.