NFAT5 is the main regulator of an osmoprotective gene program that is switched on by osmotic stress. Hypertonicity causes a harmful increase in the intracellular concentration of inorganic ions and NFAT5-regulated genes allow the accumulation of organic osmolytes and chaperones in order to protect the cells. However, little is known about the physiopathologic tonicity thresholds that trigger NFAT5 transcriptional activity, and which signaling pathways are involved, in primary cells. We have studied the regulation of NFAT5 transcriptional activity in several types of primary cells obtained from transgenic mice carrying the 9xNFAT-Luc reporter developed by the Molkentin laboratory. Our results indicate that NFAT5 is able to respond to pathological tonicity levels in primary cells and it requires a combination of signaling mediators, some of which are more relevant in specific cell types.