Primordium formation of Agaricus bisporus depends on the presence of a casing layer containing stimulatory bacteria and on sufficient air exchange. The influence of specific pseudomonad populations and volatile organic compounds (VOC) on primordium formation of A. bisporus was studied in microcosm cultures. VOC produced by A. bisporus mycelium were predominantly C8 compounds, some of which could inhibit primordium formation, with 1-octen-3-ol being most inhibitory. A VOC produced by the rye grain substrate, 2-ethyl-1-hexanol, on which A. bisporus was grown also inhibited primordium formation. 2-Ethyl-1-hexanol and 1-octen-3-ol were metabolized by pseudomonad populations and adsorbed by activated charcoal, with both modes of removal enabling primordium formation in the casing. Removal of VOC by ventilation also enabled primordium formation to occur under axenic conditions. The presence of 2-ethyl-1-hexanol and 1-octen-3-ol in the microcosms resulted in higher total bacterial and pseudomonad populations in the casing. The stimulatory effects of the casing and its microbiota and air exchange on primordium formation of A. bisporus at least partly are due to the removal of inhibitory C8 compounds produced by the mycelium and its substrate. Monitoring and controlling the levels of these inhibitory VOC in mushroom culture should enable primordium formation of A. bisporus to be more efficiently and precisely controlled.