The Knights of St. John are a campaign civilization that is only available for play in either the story mode of Age of Empires III or custom scenarios made from the Scenario Editor.
The Knights of St. John, though based in Malta, are in many ways very similar to the Spanish civilization because they have similar architectural styles, units, and Home City but speak the English language. Thus The Knights of St. John are essentially a mix of the two.
The Knights of St. John are minor opponents in one of the battles of the Saladin campaign in Age of Empires II. There, they were known as the Knights Hospitallers, and were represented by the Teuton civilization.