Clinically available anticonvulsant drugs fail to control seizures in approximately 30% of epileptic patients. If hippocampal sclerosis is combined with focal dysplasia or similar developmental alterations, the likelihood of incomplete seizure control may reach >90%. Because only a minority of epilepsy patients benefit from epilepsy surgery, we need more research into the mechanisms of drug refractoriness. In this review we analyze different approaches to study pharmacoresistance and underlying mechanisms using in vitro models. Epileptiform discharges after prolonged application of low Mg2+ artificial cerebrospinal fluid (ACSF) or 4-aminopyridine (4-AP), or combined application of these convulsants with bicuculline in acute hippocampal–entorhinal cortex slices reveal pharmacoresistance and point to loss of γ-aminobutyric acid (GABA)ergic function, in part due to reduced delivery of GABA from presynaptic terminals. Interestingly, epileptiform activity in immature tissue (organotypic hippocampal slice cultures and acute intact hippocampus) is immediately resistant to available antiepileptic drugs, and preliminary evidence points to a role of alterations in Cl− homeostasis. Seizure-like events can also be induced in dissected tissues from human epileptic patients. Future research on human tissue may provide useful information for understanding the mechanisms underlying pharmacoresistance.