Although many techniques can be used to generate multitype-induced pluripotent stem (iPS) cells from multitype seed cells, improving the efficiency and shortening the period of cell reprogramming remain troublesome issues. In this study, to generate iPS cells, CD34⁺ cells, isolated from human amniotic fluid cells (HuAFCs) by flow cytometry, were infected with retroviruses carrying only one reprogramming factor (Oct4) and cultured on human amniotic epithelial cell (HuAEC) feeder layers. Approximately 4 to 5 days after viral infection, some embryonic stem cell (ESC)-like colonies appeared among the feeder cells. These colonies were positive for alkaline phosphatase and expressed high levels of ESC pluripotent markers (Nanog, Sox2, Oct4, CD133, and Rex1). Moreover, these iPS cells exhibited high levels of telomerase activity and had normal karyotypes. Additionally, these cells could differentiate into cell types from all 3 germ layers in vivo and in teratomas. In summary, we report a novel way of iPS generation that uses CD34⁺ HuAFCs as seed cells. Using this method, we can generate human iPS cells with greater efficiency and safety (the oncogenic factors, c-Myc and Klf4, were not used), and using the minimum number of reprogramming factors (only one factor, Oct4). Besides, HuAECs were used as feeder layers to culture human iPS cells, which could not only avoid contamination with heterogeneous proteins, but also maintain iPS cells in a self-renewing and undifferentiated state for a long time.