It is well established that CNS midline cells are essential for the identity determination, division, and differentiation of neurons and glia in the Drosophila CNS. However, it is not clear whether CNS midline cells control the establishment and differentiation of the well known RP2 motoneuron lineage. The present study showed by using several RP2 lineage markers that CNS midline cells and Egfr signaling genes are required for identity determination and formation of precursors of the RP2 motoneurons. Overexpression and ectopic expression of sim and components of the EGFR signaling pathway in the ventral neuroectoderm induced the formation of extra RP2s and their sibling cells by activating EGFR signaling. We demonstrated that CNS midline cells and Egfr signaling genes play essential roles in the establishment of the RP2 motoneuron lineage. (C) 2009 Elsevier Inc. All rights reserved.