Striatal acetylcholine (ACh) has been linked to behavioral flexibility. A key component of flexibility is down-regulating responding as valued cues and actions become decoupled from positive outcomes. We used array fiber photometry in mice to investigate how ACh release across the striatum evolves during learning and extinction of Pavlovian associations. Changes in multi-phasic release to cues and consummatory actions were bi-directional and region-specific. Following extinction, increases in cue-evoked ACh release emerged in the anterior dorsal striatum (aDS) which preceded a down-regulation of anticipatory behavior. Silencing ACh release from cholinergic interneurons in the aDS blocked behavioral extinction. Dopamine release dipped below baseline for down-shifted cues, but glutamate input onto cholinergic interneurons did not change, suggesting an intrastriatal mechanism for the emergence of ACh increases. Our large-scale mapping of striatal ACh dynamics during learning pinpoints region-specific elevations in ACh release positioned to down-regulate behavior during extinction, a central feature of flexible behavior.