Human adipose tissue has proven to be an abundant, accessible, and rich source of adult mesenchymal stromal cells, suitable for tissue engineering and regenerative medicine. However, a major complication in fully investigating these cells may derive from their limited life span. Although methods to isolate, expand, and immortalize these cells have been widely reported in the literature, exhaustive explanations on the problems that can be encountered during these processes and how these can be solved have never been described. It is of fundamental importance to follow a common protocol to achieve reliable and reproducible results. Here, we describe a protocol to isolate and expand human adipose stromal cells from specimens obtained from tissue biopsies and liposuction surgical interventions. Finally, we broadly describe the cell immortalization technique, and particular attention is paid to some of the apparently “secondary” aspects.