While the regulation of the neurogenesis and oligodendrogenesis by microRNAs has been intensively studied, little is known about the role of microRNAs (miRNAs) in the development of astrocytes. Here, we report that microRNAs play an essential role in the differentiation and maturation of white matter astrocytes in mouse spinal cord tissues. In glial fibrillary acidic protein (GFAP)/Dicer conditional mutants, the initial generation of astrocyte progenitor cells was normal in the spinal cord. However, there was a much reduced number of GFAP+ astrocytes with shorter processes in the white matter of mutant tissues. In contrast, the expression of gray matter protoplasmic astrocyte marker was not affected. Together, our studies indicated that miRNAs are required for the differentiation and morphological maturation of white matter fibrous astrocytes in the developing spinal cord.