Carbon dioxide (CO2) is considered to be an important factor during incubation of eggs. Effects attributed to higher CO2 concentrations during experiment might be due to confounding effects of other environmental conditions, such as incubation temperature. To disentangle effects of eggshell temperature (EST) and CO2 concentration, an experiment was conducted. A total of 630 Cobb 500 hatching eggs from 37 to 45 wk commercial breeder flocks were collected and incubated according to treatments. The experiment was setup as a complete randomized 2 × 3 factorial design, resulting in 6 treatments. From day 8 of incubation onward, broiler eggs were exposed to one of two EST (37.8 or 38.9 °C) and one of three CO2 concentrations (0.1, 0.4 or 0.8%). Eggs were incubated in climate-respiration chambers and metabolic heat production was determined continuously. At day 18 of incubation and at 6 h after hatching, embryo and chicken quality were determined by evaluation of organ weights, navel condition, blood metabolites and hepatic glycogen. Hatching time and chicken length at 6 h after hatching showed an interaction between EST and CO2 concentration (both P = 0.001). Furthermore, no effect of CO2 concentration was found on embryo development or chicken quality. Metabolic heat production between day 8 and 18 of incubation was not affected by either EST or CO2. At day 18 of incubation, an EST of 38.9 °C resulted in a higher egg weight loss, longer embryos, higher yolk free body mass (YFBM) and lower heart weight than an EST of 37.8 °C (all P < 0.008). At 6 h after hatching, an EST of 38.9 °C resulted in a higher residual yolk weight and lower YFBM, liver weight and heart weight than an EST of 37.8 °C (all P < 0.003). Lactate, uric acid and hepatic glycogen were not affected by EST at either day 18 of incubation or at hatch. Glucose was not affected by EST at day 18 of incubation, but at hatch, it was higher at an EST of 37.8 °C than at an EST of 38.9 °C (P = 0.02). It can be concluded that effects of CO2 concentration (at concentrations ≤0.8%) on embryonic development and chicken quality appear to be limited when EST is maintained at a constant level. Moreover, a higher EST from day 8 of incubation onward appears to negatively affect chicken quality at hatch.