The increasing appreciation of the enormous developmental potential of stem cells has raised hopes that one day they can be manipulated in vitro to generate desired cell populations for clinical applications. To provide a quantitative understanding of cytokine effects that affect the maintenance of hematopoietic stem cell (HSC) function in vitro, changes in HSC numbers in serum-free cultures were evaluated with a modified competitive repopulation unit (CRU) assay based on the level of donor-derived engraftment. A factorial design was used to screen the effects of thrombopoietin, flt-3 ligand (FL), Steel factor (SF), interleukin (IL)-11, incubation temperature and hydrocortisone in cultures of Sca-1⁺lin⁻ mouse bone marrow. The results indicated that IL-11 and SF were the most important stimulators of CRU expansion. When response surface methodology was used to analyze cytokine effects, IL-11 was found to have a bell-shaped dose-response with a maximum effect at 20 ng/mL. High concentration saturation or decrease in the effect of SF or FL was not observed and their stimulatory effect increased continuously, beyond 300 ng/mL. A negative interaction between SF and FL was also discovered that resulted in a significant inhibitory effect when both were added to cultures at high concentrations (>100 ng/mL). When the response of CRUs was compared with colony-forming cells and total cells, a surprisingly similar pattern of cytokine effects was found. However, the populations differed dramatically in their responsiveness to cytokine doses such that the maintenance of CRU numbers in vitro required > 50-fold greater concentrations. The observed pattern of cytokine effects guided the development of a mathematical model of cell expansion based on Monod kinetics. The HSC proliferation and self-renewal response requirements for gpl30 activation were also examined in single-cell cultures. HSC survival and mitogenesis were maximized by exposure to SF and FL, but optimal retention of their functional capacity required the additional presence of a cytokine that will activate gpl30. Taken together, these results have demonstrated that HSCs mitogenesis and self-renewal can be independently manipulated by the activation of different cell surface cytokine receptors, with additional differential effects dictated by the intensity of signaling.