More than 10,000 IEQ/kg recipient weight of islets is often necessary to achieve insulin independence in patients with type 1 diabetes mellitus. Several studies have identified high donor body mass index (BMI) and pancreas size as important factors for the success of human islet isolation. However, the donor shortage underscores the need to improve isolation outcomes from lower BMI pancreas donors and/or small pancreata. The aim of this study was to identify the critical factors that affect isolation outcome. We analyzed the data from 207 isolations performed from 2002 to 2006 with respect to donor characteristics, pancreas condition, and processing variables. More than 3000 IEQ/g pancreas weight was considered to be an acceptable isolation outcome. This goal was obtained from donors with a BMI >30 kg/m2 (P = .002). The pancreatic surface integrity was also a significant factor (P = .02). Moreover, longer digestion times (P = .04) and a greater proportion of trapped islets negatively affected success rates (P = .004). As previously reported, pancreata from high BMI donors were suitable for islet isolation and transplantation, as they yielded higher total islet particle numbers and higher IEQ/g. Although BMI and pancreas size are not controllable due to the organ donor shortage, factors such as pancreatic surface integrity, shorter digestion time, and lower proportions of trapped islets were found to be significant to obtain higher success rates. The development of better protocols and systematic training of processing/procurement teams will be of assistance to increase the number of successful human islet isolations.