The phytopathogenic actinomycete Rhodococcus fascians D188 relies mainly on the linear plasmid-encoded fas operon for its virulence. The bacteria secrete six cytokinin bases that synergistically redirect the developmental program of the plant to stimulate proliferation of young shoot tissue, thus establishing a leafy gall as a niche. A yeast-based cytokinin bioassay combined with cytokinin profiling of bacterial mutants revealed that the fas operon is essential for the enhanced production of isopentenyladenine, trans-zeatin, cis-zeatin, and the 2-methylthio derivatives of the zeatins. Cytokinin metabolite data and the demonstration of the enzymatic activities of FasD (isopentenyltransferase), FasE (cytokinin oxidase/dehydrogenase), and FasF (phosphoribohydrolase) led us to propose a pathway for the production of the cytokinin spectrum. Further evaluation of the pathogenicity of different fas mutants and of fas gene expression and cytokinin signal transduction upon infection implied that the secretion of the cytokinin mix is a highly dynamic process, with the consecutive production of a tom initiation wave followed by a maintenance flow.