Starvation induces cell aggregation in the soil bacterium Myxococcus xanthus, followed by formation of fruiting bodies packed with myxospores. Sporulation in the absence of fruiting bodies can be artificially induced by high concentrations of glycerol through unclear mechanisms. Here, we show that a compound (ambruticin VS-3) produced by a different myxobacterium, Sorangium cellulosum, affects the development of M. xanthus in a similar manner. Both glycerol (at millimolar levels) and ambruticin VS-3 (at nanomolar concentrations) inhibit M. xanthus fruiting body formation under starvation, and induce sporulation in the presence of nutrients. The response is mediated in M. xanthus by three hybrid histidine kinases (AskA, AskB, AskC) that form complexes interacting with two major developmental regulators (MrpC, FruA). In addition, AskB binds directly to the mrpC promoter in vitro. Thus, our work indicates that the AskABC-dependent regulatory pathway mediates the responses to ambruticin VS-3 and glycerol. We hypothesize that production of ambruticin VS-3 may allow S. sorangium to outcompete M. xanthus under both starvation and growth conditions in soil.