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Here a genome-wide RNAi library screen is used to identify components of the signalling pathway that allow transformation of Trypanosoma brucei spp., the protozoan parasite responsible for important human and livestock diseases in sub-Saharan Africa, from proliferative slender forms to arrested stumpy forms which are transmitted to the tsetse fly vector. Trypanosoma brucei spp., the protozoan parasites responsible for human African trypanosomiasis and the livestock disease nagana in sub-Saharan Africa, proliferate as slender forms in the mammalian bloodstream then differentiate into to stumpy forms that are transmitted to the tsetse fly vector. This differentiation is regulated by a quorum-sensing-like process that depends on the 'stumpy induction factor' and a downstream signalling pathway, whose respective identity and competition have proven elusive. Here Keith Matthews and colleagues have used a genome-wide RNA interference library screen to identify the components of the signalling pathway, from purine metabolism, through signal transducers (kinases, phosphatases) to gene expression regulators. The identified molecules could be possible targets for quorum-sensing interference, a strategy with antimicrobial potential against bacterial pathogens. The protozoan parasites Trypanosoma brucei spp. cause important human and livestock diseases in sub-Saharan Africa. In mammalian blood, two developmental forms of the parasite exist: proliferative ‘slender’ forms and arrested ‘stumpy’ forms that are responsible for transmission to tsetse flies. The slender to stumpy differentiation is a density-dependent response that resembles quorum sensing in microbial systems and is crucial for the parasite life cycle, ensuring both infection chronicity and disease transmission1. This response is triggered by an elusive ‘stumpy induction factor’ (SIF) whose intracellular signalling pathway is also uncharacterized. Laboratory-adapted (monomorphic) trypanosome strains respond inefficiently to SIF but can generate forms with stumpy characteristics when exposed to cell-permeable cAMP and AMP analogues. Exploiting this, we have used a genome-wide RNA interference library screen to identify the signalling components driving stumpy formation. In separate screens, monomorphic parasites were exposed to 8-(4-chlorophenylthio)-cAMP (pCPT-cAMP) or 8-pCPT-2′-O-methyl-5′-AMP to select cells that were unresponsive to these signals and hence remained proliferative. Genome-wide Ion Torrent based RNAi target sequencing identified cohorts of genes implicated in each step of the signalling pathway, from purine metabolism, through signal transducers (kinases, phosphatases) to gene expression regulators. Genes at each step were independently validated in cells naturally capable of stumpy formation, confirming their role in density sensing in vivo. The putative RNA-binding protein, RBP7, was required for normal quorum sensing and promoted cell-cycle arrest and transmission competence when overexpressed. This study reveals that quorum sensing signalling in trypanosomes shares similarities to fundamental quiescence pathways in eukaryotic cells, its components providing targets for quorum-sensing interference-based therapeutics.