The co-culture of human epidermal keratinocytes with mouse 3T3-J2 feeder cells, a method developed by Green and colleagues, has been widely used since the early 1980s to generate skin autografts. Additionally, co-culture with 3T3-J2 cells has been a crucial tool in skin stem cell biology, as it facilitates the evaluation of self-renewal capacity and differentiation of epidermal stem cells. This chapter presents a recent enhancement of the Green method, aimed at further promoting the expansion of human epidermal keratinocytes by utilizing a small molecule inhibitor of TGF-β signaling. This new protocol also enables the rapid expansion of human epidermal keratinocytes in co-culture with human feeder cells, including human dermal fibroblasts and human preadipocytes—two key alternatives to 3T3-J2 cells—with the long-term goal of developing customized Xeno-free skin autografts.