Measurement of soil respiration to quantify ecosystem carbon cycling requires absolute, not relative, estimates of soil CO2 efflux. We describe a novel, automated efflux apparatus that can be used to test the accuracy of chamber-based soil respiration measurements by generating known CO2 fluxes. Artificial soil is supported above an air-filled footspace wherein the CO2 concentration is manipulated by mass flow controllers. The footspace is not pressurized so that the diffusion gradient between it and the air at the soil surface drives CO2 efflux. Chamber designs or measurement techniques can be affected by soil air volume, hence properties of the soil medium are critical. We characterized and utilized three artificial soils with diffusion coefficients ranging from 2.7 × 10−7 to 11.9 × 10−7 m2 s−1 and porosities of 0.26 to 0.46. Soil CO2 efflux rates were measured using a commercial dynamic closed-chamber system (Li-Cor 6400 photosynthesis system equipped with a 6400-09 soil CO2 flux chamber). On the least porous soil, small underestimates (< 5%) of CO2 effluxes were observed, which increased as soil diffusivity and soil porosity increased, leading to underestimates as high as 25%. Differential measurement bias across media types illustrates the need for testing systems on several types of soil media.