<p>Preterm or complicated labor is often accompanied by the asphyxia of the newborn. Temporal limitation of oxygen and nutrient supply can be especially harmful to the developing brain and lead to hypoxic-ischemic encephalopathy. This injury is mainly caused by the reduction in white matter volume and may be triggered by delayed maturation of oligodendrocytes and affected myelination. One of the key factors involved in the pathophysiology of neural tissue after asphyxia is the activation of hypoxia-inducible factor 1α (HIF-1α) signaling. The objective of the study was to verify whether this factor is involved in affected oligodendrocyte differentiation in this injury and whether its modulation can be considered as a treatment of hypoxic-ischemic encephalopathy. In the study, we mimicked the hypoxic-ischemic injury to <i>in vitro</i> cultured oligodendrocyte progenitor cells obtained from rat pups. Cells were deprived of culture medium and exposed to 50&#xa0;min of oxygen deficit. After the insult, an enhanced activation (CoCl₂) or chemical inhibition (KC7F2) of HIF-1α was induced. We analyzed cell viability, proliferation, oligodendrocyte maturation, and the expression of myelin proteins. The injury did not cause massive oligodendrocyte progenitor death; neither any of applied treatments exerted cytotoxic effect. HIF-1α inhibition contributed to enhanced oligodendrocyte maturation. Continuing hypoxia stimulation with CoCl₂ was associated with an increase in the number of oligodendrocytes, which were, however, less differentiated. These findings suggest an important role for HIF-1α signaling in oligodendrocyte differentiation. Further studies could help to define a new target for the therapy of hypoxic-ischemic encephalopathy.</p>