The Chesapeake 2000 Agreement committed its state and federal signatories to “define the water quality conditions necessary to protect aquatic living resources” in the Chesapeake Bay (USA) and its tidal tributaries. Hypoxia is one of the key water quality issues addressed as a result of the above Agreement. This paper summarizes the protection goals and specific criteria intended to achieve those goals for addressing hypoxia. The criteria take into account the variety of Bay habitats and the tendency towards low dissolved oxygen in some areas of the Bay. Stressful dissolved oxygen conditions were characterized for a diverse array of living resources of the Chesapeake Bay by different aquatic habitats: migratory fish spawning and nursery, shallow-water, open-water, deep-water, and deep-channel. The dissolved oxygen criteria derived for each of these habitats are intended to protect against adverse effects on survival, growth, reproduction and behavior. The criteria accommodate both spatial and temporal aspects of low oxygen events, and have been adopted into the Chesapeake Bay states – Maryland, Virginia, and Delaware – and the District of Columbia's water quality standards regulations. These criteria, now in the form of state regulatory standards, are driving an array of land-based and wastewater pollution reduction actions across the six-watershed.