Freeze-drying is a common method for preservation of probiotics, including bifidobacteria, for further industrial applications. However, the stability of freeze-dried bifidobacteria varies depending on the freeze-drying method and subsequent storage conditions. The primary goals of this study were to develop an optimized freeze-drying procedure and to determine the effects of temperature, water activity, and atmosphere on survival of freeze-dried bifidobacteria. To address these goals, a commercially used bifidobacteria strain that is resilient to stress, Bifidobacterium animalis ssp. lactis Bb-12, and a characterized intestinal strain that is more sensitive to stress conditions, Bifidobacterium longum DJO10A, were used. A freeze-drying protocol was developed using trehalose as the cryoprotectant, which resulted in almost no loss of viability during freeze-drying. Resuscitation medium, temperature, and time did not significantly influence recovery rates when this cryoprotectant was used. The effects of temperature (-80 to 45°C), water activity (0.02 to 0.92), and atmosphere (air, vacuum, and nitrogen) were evaluated for the stability of the freeze-dried powders during storage. Freeze-dried B. animalis ssp. lactis Bb-12 was found to survive under all conditions tested, with optimum survival at temperatures up to 21°C, water activities up to 0.44, and all 3 atmospheres tested. The intestinal-adapted strain B. longum DJO10A was much more sensitive to the different storage conditions, but could be adequately maintained using optimum conditions. These optimum storage conditions included frozen storage, replacement of oxygen with nitrogen, and water activities between 0.11 and 0.22. These results indicated that an optimized storage environment is required to maintain viability of stress-sensitive bifidobacteria strains, whereas stress-resilient bifidobacteria strains can maintain viability over a wide range of storage conditions, which is practical in countries where controlled cold storage conditions may not be readily available.