Many cellular therapies require a cryopreservation step during their lifecycle. Cryopreservation offers many advantages such as extending product shelf life, decoupling manufacture from “just-in-time” delivery, and allowing pre-conditioning and other treatment interventions before administration. Nevertheless, it can also pose significant risks to the product and by extension the patient, if the process is non-optimized or misapplied. A clear understanding of the principles and their relationship to cell viability and functionality is therefore a pre-requisite for effective cryopreservation. The effect of cooling rate on cell survival, the need to avoid the formation of intracellular ice, and the action of cryoprotectants in improving cell recovery are all well recognized. Less so, the need to control cryoprotectant exposure times/temperature and their part in controlling cryoprotectant toxicity and the avoidance during addition/removal of damaging excursions in cell volume. However, there are other, newly emerging, factors that often go unrecognized. This chapter sets out to examine some that are potential impediments to the successful generation of a cell therapy product. Recently, a debate has opened up on the use of dimethyl sulphoxide as the cryoprotectant of choice in the context of cell therapies. Its use, together with that of bovine serum as an excipient, is being questioned and an examination of the advantages, disadvantages, and alternatives is explored herein. The control of ice nucleation/propagation and the phenomenon of supercooling which, if uncontrolled, can lead to excessive ice nucleation and variable outcomes, and the means to control this process and inhibit ice crystallization are also discussed. The effect on cell viability of transient warming events during routine low-temperature storage has, until recently, gone unrecognized. This is an emerging issue with both regulatory and commercial implications. Lastly, cryopreservation-induced delayed-onset cell death, its causes, detection and prevention, and the implications of such injury on demonstrating acceptable viability and functionality for regulatory purposes and routine quality-control monitoring are also examined.