Ischemia has elicited a great deal of interest among the scientific community due to its role in life-threatening pathologies such as cancer, stroke, acute renal failure, and myocardial infarction. Oxygen deprivation (hypoxia) associated with ischemia has recently become a subject of intense scrutiny. New investigators may find it challenging to induce hypoxic injury in vitro. Researchers may not always be aware of the experimental barriers that contribute to this phenomenon. Furthermore, ischemia is associated with other major insults, such as excess carbon dioxide (hypercapnia), nutrient deprivation, and accumulation of cellular wastes. Ideally, these conditions should also be incorporated into in vitro models. Therefore, the motivation behind this review is to: i. delineate major in vivo ischemic insults; ii. identify and explain critical in vitro parameters that need to be considered when simulating ischemic pathologies; iii. provide recommendations to improve experiments; and as a result, iv. enhance the validity of in vitro results for understanding clinical ischemic pathologies. Undoubtedly, it is not possible to completely replicate the in vivo environment in an ex vivo model system. In fact, the primary goal of many in vitro studies is to elucidate the role of specific stimuli during in vivo pathological events. This review will present methodologies that may be implemented to improve the applicability of in vitro models for understanding the complex pathological mechanisms of ischemia. Finally, although these topics will be discussed within the context of renal ischemia, many are pertinent for cellular models of other organ systems and pathologies.