Islet transplantation is emerging as a viable treatment option for selected patients with type 1 diabetes. Following the initial report in 2000 from Edmonton of insulin independence in seven out of seven consecutive recipients, there has been a huge expansion in clinical islet transplantation. The challenge we now face is the apparent decline in graft function over time. Isolating high-quality human islets which survive and function for a longer period will no doubt contribute to further improvement in long-term clinical outcome. This chapter reviews the selection of appropriate donors for islet isolation and transplantation, describes each step during islet isolation, and discusses the scope for further improvements.