Fungal propagules survive stresses better than vegetative cells. Neosartorya fischeri, an Aspergillus teleomorph, forms ascospores that survive high temperatures or drying followed by heat. Not much is known about maturation and development of extreme stress resistance in fungal cells. This study provides a novel two-step model for the acquisition of extreme stress resistance and entry into dormancy. Ascospores of 11- and 15-day-old cultures exhibited heat resistance, physiological activity, accumulation of compatible solutes and a steep increase in cytoplasmic viscosity. Electron spin resonance spectroscopy indicated that this stage is associated with the removal of bulk water and an increase of chemical stability. Older ascospores from 15- to 50-day-old cultures showed no changes in compatible solute content and cytoplasmic viscosity, but did exhibit a further increase of heat resistance and redox stability with age. This stage was also characterized by changes in the composition of the mixture of compatible solutes. Mannitol levels decreased and the relative quantities of trehalose and trehalose-based oligosaccharides increased. Dormant ascospores of N. fischeri survive in low-water habitats. After activation of the germination process, the stress resistance decreases, compatible solutes are degraded and the cellular viscosity drops. After 5 h, the hydrated cells enter the vegetative stage and redox stability has decreased notably.