A hallmark of tuberculosis (TB) is the presence of tubercles, which are granulomas composed of activated alveolar macrophages, neutrophils and lymphocytes. Tubercles are repositories for the causative agent of TB, Mycobacterium tuberculosis, and are partly generated by virulence factors encoded by the bacterium. Using a close relative of M. tuberculosis, Mycobacterium marinum, Stoop et al. develop a medium-throughput screen for virulence factors using M. marinum’s natural host, the zebrafish, and identify three novel virulence factors that are involved in granuloma formation. This system provides new information about mycobacterial virulence and might help in the development of new TB vaccines.