Cognitive flexibility is the capability to appropriately adapt one's behavior in response to new and unexpected conditions in the environment (Prado et al., Neuroscience, 2017). This cognitive function plays a fundamental role in our ability to adapt and learn. When cognitive flexibility encompasses spatial information, it is supported by a hippocampo-cortico-thalamic network in which the reuniens and rhomboid nuclei (ReRh) of the thalamus may relay information between hippocampus and the medial prefrontal cortex (mPFC). We showed that reversible inactivation (muscimol or DREADD for ReRh) of one of these structures produces a marked strategy shifting deficit in a water maze escape navigation task for rodents in the Double-H maze (e.g., Cholvin et al., J Neurosci, 2013). To further our understanding of this network, we induced a definitive yet projection-specific lesion of the cortico-thalamic (from mPFC to ReRh) pathway in rats. For this, we used a viral approach allowing the expression of a caspase in the mPFC under Cre-recombinase dependency coming from the ReRh. We demonstrated a disruption of about 70% of the retrogradely labelled mPFC neurons projecting to the ReRh. Behavioral analysis showed that disconnected rats had lost the ability to switch from an egocentric to an allocentric navigation strategy when modified task contraints required it, indicating a deficit in cognitive flexibility. These data clearly show that mPFC neurons projecting to the ReRh play a major role in cognitive flexibility.