Mouse and human somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by the transduction of four transcription factors, Oct 3/4, Sox2, Klf4, and c-Myc (Maherali et al., 2007; Meissner et al., 2007; Okita et al., 2007; Takahashi et al., 2007; Takahashi and Yamanaka, 2006; Wernig et al., 2007). Patient or disease-specific human iPSCs could be used for studying pathogenesis, or potentially also to treat patients suffering from incurable diseases by transplanting the regenerated grafts derived from their own cells. However, the low induction efficiency and high tumorigenesis rate due to the use of proto-oncogenes, such as c-Myc, continue to hinder the clinical application of iPS technology. Many efforts have been made to find other factors or small molecules that facilitate the reprogramming process (Huangfu et al., 2008; Shi et al., 2008b). In this study, we show that conducting reprogramming in hypoxic conditions results in improved efficiency for both mouse and human cells. Somatic stem cells reside in specific microenvironments, called niches, and environmental changes, such as stromal cell contacts, extracellular matrix proteins, temperature, and O2 tension, have a great influence on stem cell function and differentiation. Notably, low O2 tension promotes the survival of neural crest cells and hematopoietic stem cells and prevents differentiation of human ESCs (Danet et al., 2003; Ezashi et al., 2005; Morrison et al., 2000). Moreover, mammalian embryonic epiblasts reside in a physiologically hypoxic environment. These observations led us to the hypothesis that hypoxic conditions might promote the reprogramming process and thus iPS cell generation. To quantify the effect of hypoxia on iPS cell generation, we performed comparison experiments on mouse embryonic fibroblasts (MEFs) carrying the Nanog-GFP-Ires-Puror cassette (Okita et al., 2007). Four or three transcription factors (Oct3/4, Sox2, Klf4,+/À c-Myc) were introduced into MEFs with retroviral vectors. Four days after transduction, the cells were trypsinized and seeded onto the feeder layer of mitomycin C-treated STO cells. The cells were cultivated under 21%, 5%, or 1% O2 from day 5 to day 14 after transduction. GFP+ iPS cell colonies could be first detected on days 10–14 after viral transduction, and we counted the number of GFP-positive colonies on days 21 and 28 after transduction. Under 5% O2, the GFP-positive colonies derived from four-factor transduced MEFs increased 7.4-fold on day 21 and 3.1-fold on day 28, and those from three-factor transduced MEFs increased 20-fold on day 21 and 7.6-fold on day 28 under 5% O2 (Figures 1 Aa and 1Ab). Moreover, hypoxic treatment increased the percentage of GFP-positive colonies in total colonies from four-or three-factor transduced MEFs (Figures 1 Ac and 1Ad). The GFP-positive colonies derived after hypoxic treatment was comparable in morphology and size to those derived under normoxic conditions (Figure S1 available online). Alkaline phosphatase staining showed that cultivation under 5% O2 increased the number of colonies with a positive alkaline phosphatase activity (Figure S2). We also examined whether GFP-positive cells were detected more quickly under hypoxic conditions. The four-factor transduced MEFs were cultivated under 21% O2 or under 5% O2 with or without VPA from day 5 to day 9 after transduction and were subjected to flow cytometric analysis on day 9. Retroviral expression of four factors induced 0.01% of the cells to become GFP-positive on day 9 after transduction. Treating the four-factor transduced MEFs for 4 days with hypoxia or with VPA increased the percentage of GFP-positive cells …