The dorsal striatum receives converging excitatory inputs from diverse brain regions, including the cerebral cortex and the intralaminar/midline thalamic nuclei, and mediates learning processes contributing to instrumental motor actions. However, the roles of each striatal input pathway in these learning processes remain uncertain. We developed a novel strategy to target specific neural pathways and applied this strategy for studying behavioral roles of the pathway originating from the parafascicular nucleus (PF) and projecting to the dorsolateral striatum. A highly efficient retrograde gene transfer vector encoding the recombinant immunotoxin (IT) receptor was injected into the dorsolateral striatum in mice to express the receptor in neurons innervating the striatum. IT treatment into the PF of the vector-injected animals caused a selective elimination of neurons of the PF-derived thalamostriatal pathway. The elimination of this pathway impaired the response selection accuracy and delayed the motor response in the acquisition of a visual cue-dependent discrimination task. When the pathway elimination was induced after learning acquisition, it disturbed the response accuracy in the task performance with no apparent change in the response time. The elimination did not influence spontaneous locomotion, methamphetamine-induced hyperactivity, and motor skill learning that demand the function of the dorsal striatum. These results demonstrate that thalamostriatal projection derived from the PF plays essential roles in the acquisition and execution of discrimination learning in response to sensory stimulus. The temporal difference in the pathway requirement for visual discrimination suggests a stage-specific role of thalamostriatal pathway in the modulation of response time of learned motor actions.