The long-term repopulating capacity of mobilized peripheral blood (MPB) CD34+ cells can be abrogated during ex vivo expansion due to cell-cycle related changes. In this study, we traced cell cycle kinetic and quiescent status of CD34+ cells during ex vivo expansion with exogenous cytokines. The addition of interleukin-3 (IL-3) and interleukin-6 (IL-6) to early acting cytokines resulted in a significantly lower percentage of cells remaining undivided or in quiescent G0 phase when compared to the use of early acting cytokines alone. We conclude that ex vivo expansion of MPB CD34+ cells with a cocktail of five cytokines yields a highly cycling offspring, which could impair long-term repopulating capacity.