The stomatal response of seedlings grown in 360 or 720 μmol mol−1 to irradiance and leaf-to-air vapour pressure deficit (VPD) at both 360 and 720 μmol mol−1 to CO2 was measured to determine how environmental factors interact with CO2 enrichment to affect stomatal conductance. Seedlings of four species with different conductances and life histories, Cercis canadensis (L.), Quercus rubra (L.), Populus deltoides (Bartr. ex Marsh.) × P. nigra (L.), and Pinus taeda (L.), were measured in hopes of identifying general responses. Conductance of seedlings grown at 360 and 720 μmol mol−1 CO2 were similar and responded in the same manner to measurement CO2 concentration, irradiance and VPD. Conductance was lower for all species when measured at 720 than when measured at 360 μmol mol−1 CO2 at both VPDs (˜1.5 and ˜2.5 kPa) and all measured irradiances greater than zero (100, 300, 600,>1600 μmol m−2 S−2) The average decrease in conductance due to measurement in elevated CO2 concentration was 32% for Cercis, 29% for Quercus, 26% for Populus, and 11% for Pinus. For alt species, the absolute decrease in conductance due to measurement in CO2 enrichment decreased as irradiance decreased or VPD increased. The proportional decrease due to measurement in CO2 enrichment decreased in three of eight cases: from 0.46 to 0.10 in Populus and from 0.18 to 0.07 in Pinus as irradiance decreased from>1600 to 100 μmol m−2 s−1 and from 0.35 to 0.24 in Cercis as VPD increased from 1.3 to 2.6 kPa.