Rhomboid (Rho), a cell surface, seven-transmembrane domain protein, participates in Spitz-dependent activation of the Drosophila EGF receptor (EGFR). By contrast to transient expression in other embryonic tissues, rho is expressed continuously in the embryonic and larval Midline Glia (MG) lineage and is required upstream of, or in parallel with, S, Spi, and EGFR to establish MG cell number. EGFR signaling is necessary for the expression of rho in the MG and sufficient to stimulate rho expression in additional MG progenitors. rho expression is required continuously from embryonic stage 9-17 to suppress apoptosis in the MG. Although rho misexpression can increase MG number through a non-cell autonomous mechanism, the pattern of normal rho expression suggests that it functions by enhancing autocrine or paracrine signaling among MG cells.