Cultured keratinocytes (KC) are needed for transplantation to the surface of large burn wounds and ulcers. They can be cultured in artificial media. However, the yield is always limited, viability is low, and proliferation and migration after grafting are slow. The question arose whether tissue fluid/lymph, which is a natural humoral environment for epidermal and dermal cells, contains cytokine(s) specifically regulating KC proliferation and could be used to culture large numbers of cells for transplantation. Culturing of skin keratinocytes in dermal tissue fluid/lymph containing keratinocyte growth factor, interleukin-1beta, interleukin-6, tumor necrosis factor-alpha, and transforming growth factor-beta revealed its strong stimulatory effect on the expression of p63 stem cell marker and proliferation but not differentiation of KC. Neutralizing these cytokines with antibodies resulted in decreased percentages of mitotic figures. None of the individual cytokines showed a dominant effect on proliferation. This observation suggests that either there may be other (so far undetected) specific cytokines or that the proliferation and differentiation of keratinocytes is an effect of the combined action of all investigated cytokines.