In general, stomatal density (SD) decreases when plants are grown at high CO2 concentrations. Recent studies suggest that signals produced from mature leaves regulate the SD of expanding leaves. To determine the underlying driver of these signals in poplar (Populus trichocarpaxP. deltoides) saplings, a cuvette system was used whereby the environment around mature (lower) leaves could be controlled independently of that around developing (upper) leaves. A series of experiments were performed in which the CO2 concentration, vapour pressure deficit (D), and irradiance (Q) around the lower leaves were varied while the (ambient) conditions around the upper leaves were unchanged. The overall objective was to break the nexus between leaf stomatal conductance and transpiration and photosynthesis rates of lower leaves and determine which, if any, of these parameters regulate stomatal development in the upper expanding leaves. SD, stomatal index (SI), and epidermal cell density (ED) were measured on the adaxial and abaxial surfaces of fully expanded upper leaves. SD and SI decreased with increasing lower leaf CO2 concentration (150-780 ppm) at both ambient (1.3-1.6 kPa) and low (0.7-1.0 kPa) D. SD and SI at low D were generally higher than at ambient D. By contrast, ED was relatively insensitive to both vapour pressure and CO2 concentration. When lower leaves were shaded, upper leaf SD, SI, and ED decreased but did not change with varying CO2 concentration. These results suggest that epidermal cell development and stomatal development are regulated by different physiological mechanisms. SI of the upper leaves was positively and highly correlated (r2>0.84) with the stomatal conductance of the lower leaves independent of their net photosynthesis and transpiration rates, suggesting that the stomatal conductance of mature leaves has a regulatory effect on the stomatal development of expanding leaves.