Mitotic spindle assembly in centrosome-containing cells relies on two main microtubule (MT) nucleation pathways, one based on centrosomes and the other on chromosomes. However, the relative role of these pathways is not well defined. In Drosophila, mutants without centrosomes can form functional anastral spindles and survive to adulthood. Here we show that mutations in the Drosophila misato (mst) gene inhibit kinetochore-driven MT growth, lead to the formation of monopolar spindles and cause larval lethality. In most prophase cells of mst mutant brains, asters are well separated, but collapse with progression of mitosis, suggesting that k-fibers are essential for maintenance of aster separation and spindle bipolarity. Analysis of mst; Sas-4 double mutants showed that mitotic cells lacking both the centrosomes and the mst function form polarized MT arrays that resemble monopolar spindles. MT regrowth experiments after cold exposure revealed that in mst; Sas-4 metaphase cells MTs regrow from several sites, which eventually coalesce to form a single polarized MT array. By contrast, in Sas-4 single mutants, chromosome-driven MT regrowth mostly produced robust bipolar spindles. Collectively, these results indicate that kinetochore-driven MT formation is an essential process for proper spindle assembly in Drosophila somatic cells.