Genetic and epigenetic factors contribute to the development of the spinal cord. Failure in correct exertion of the developmental programs, including neurulation, neural tube closure and neurogenesis of the diverse spinal cord neuronal subtypes results in clinical phenotypes with variable severity. The histone methyltransferase Disruptor of Telomeric 1 Like (DOT1L), which mediates histone H3 lysine 79 (H3K79) methylation, is fundamental for proper development of the cerebral cortex and cerebellum, and here we report on its essential role for development of the spinal cord. Conditional inactivation of DOT1L using Wnt1-cre as driver in the developing murine spinal cord did not result in neural tube closure defect (NTCD). Transcriptome analysis revealed that DOT1L deficiency favored differentiation over progenitor proliferation. Dot1l -cKO mainly decreased the numbers of dI1 interneurons expressing Lhx2 . Loss of DOT1L affected localization but not generation of dI2, dI3, and dI5 interneurons. The resulting derailed interneuron patterns might be responsible for increased cell death that occurred at the late developmental stage E18.5. Together our data indicate that DOT1L is essential for subtype- specific neurogenesis, migration and localization of interneurons in the developing spinal cord, in part by regulating transcriptional activation of Lhx2 .