Demyelinating diseases, such as multiple sclerosis, are known to result from acute or chronic injury to the myelin sheath and inadequate remyelination. Its underlying molecular mechanisms, however, remain unclear. The transcription factor prospero homeobox 1 (Prox1) plays an essential role during embryonic development of the central nervous system and cell differentiation. Thus, we aimed to investigate the role of Prox1 in the survival and differentiation of oligodendrocytes. Cell viability was measured by MTT assay. Flow cytometry was conducted to analyze cell apoptosis. Ectopic-Prox1 and shProx1 were used for the overexpression and knockdown respectively of Prox1 in FBD-102b cells. Real-time reverse transcriptase polymerase chain reaction and western blot analysis were used to assess the alterations of signaling pathway-related mRNAs and proteins, respectively. Results showed that Prox1 was upregulated in differentiating oligodendrocytes, and Prox1 knockdown inhibited the differentiation of oligodendrocytes. In addition, overexpression of Prox1 promoted oligodendrocyte differentiation, as shown by the change in myelin basic protein expression. The overexpression of Prox1 had no effect on oligodendrocyte survival, while Prox1 knockdown impaired cell survival. Further study demonstrated that Prox1 knockdown promoted oligodendrocyte apoptosis and activated NOXA, a pro-apoptotic member of the Bcl-2 protein family. Knockdown of NOXA by siRNA abrogated Prox1 knockdown-induced apoptosis. Our findings indicated that Prox1 regulated the differentiation of oligodendrocyte precursor cells via the regulation of NOXA. Therefore, Prox1 could be a potential modulator of demyelinating diseases in clinical settings.