For the ex vivo expansion of CD34(+) cells, culture conditions were optimized using cytokine cocktails and media change methods. In addition, static, orbital-shake, and stirred cultures were compared. After cultivation, total cell expansion, immunophenotypes, clonogenic ability, and metabolite concentration in media were analyzed. Optimized media change methods enhanced the number of total nucleated cells (TNCs) by 600-fold (from 10(4) to 6 × 10(6) cells) in static cultures. Furthermore, intermittent orbital-shake cultures gave the highest fold increase of TNCs and CD34(+)/CD38(-) cells. These results imply that proliferation of CD34(+) cells in intermittent shake cultures was more efficient than that in static cultures under optimized culture conditions.