The maintenance of pluripotency of human embryonic stem cells (hESCs) requires a high efficiency of self-renewal. During in vitro propagation, however, spontaneous differentiation occurs frequently, and there is also a risk of chromosomal changes. In this study, we assessed the properties of hESCs after long-term culture at ambient air and 5% oxygen growth conditions. The hESC lines were grown for up to 42 and 18 mo in normoxic and hypoxic conditions, respectively, and their proliferation; expression of Oct4, SSEA1, Nanog, and Notch1; karyotype; telomerase activity; and differentiation potential in vitro were evaluated. In contrast to cultures at 20% oxygen, where the central zones of the colonies underwent spontaneous differentiation, during exposure to 5% oxygen, the hESC colonies maintained a homogenous and flat morphology that was consistent with the presence of Oct4-positive undifferentiated phenotype. Irrespective of oxygen concentration, the undifferentiated cells expressed high levels of Nanog and Oct4 transcripts, normal karyotype, and high telomerase activity. When assayed for differentiation potential, they yielded derivatives of all three embryonic germ layers. Our data thus indicate that hypoxic exposure has the capacity to sustain enhanced long-term self-renewal of hESCs. The hESC lines described in the current paper can be obtained for research purposes from the Laboratory for Stem Cell Research, Aalborg University.