The mammalian cerebral cortex comprises deep- and upper-layer neurons (layer V-VI and II-IV, respectively), and glial cells including astrocytes. During corticogenesis, neural stem cells (NSCs) start generating deep-layer neurons at mid-gestation, then continuously change their differentiation property to produce upper-layer neurons and astrocytes. Accumulating studies unveil sophisticated mechanisms of neuron to glial fate switch of NSCs, while molecular mechanisms underlying deep- to upper-layer neuronal choice are not well understood. Oxygen is known as one of the important niche factors for SCs in various types of tissues. Although standard in vitro cultures are exposed to the environmental oxygen concentration of 21% (normoxia), actual oxygen level in the developing brains is 1-8% (hypoxia). Recently, we have shown that the acquisition of astrogenic potential by NSCs is delayed in the normoxic culture condition, yet the culture under hypoxic condition (2%) can restore this deficit (Mutoh et al. 2012). Herein, we further analyze the impact of oxygen levels in the neuronal subtype choice of NSCs. NSCs at mid-gestation that mainly produce deep-layer neurons were cultured and induced to neural differentiation under normoxic or hypoxic condition, and subsequently, generated neuronal subtypes were evaluated by immunocytochemistry. We found that hypoxia enhanced the production of Satb2-positive upper-layer neurons compared with normoxia. Knockdown of the master oxygen sensor Hif1a (hypoxia inducible factor 1a) abolished hypoxia-enhanced production of upper-layer neurons, suggesting that oxygen levels contribute to appropriate scheduling of not only neuron-glia fate switching but also neuronal subtype specification during brain development.