Spring wheat cv. Minaret was grown in open-top chambers at four sites across Europe. The effect of different treatments (CO2 enrichment, O3 fumigation, drought stress and temperature) on the chlorophyll content of the flag leaf was investigated using the MINOLTA SPAD-502 meter. Under optimum growth conditions the maximum chlorophyll content, which was reached at anthesis, was consistent among the sites ranging from 460 to 500 mg chlorophyll m−2. No significant effect of elevated CO2 or O3 was observed at anthesis. Leaf senescence, indicated by the chlorophyll breakdown after anthesis, was relatively constant in the control chambers. Under control conditions, thermal time until 50% chlorophyll loss was reached was 600°C day. Elevated CO2 caused a faster decline in chlorophyll content (thermal time until 50% chlorophyll loss was reduced to 500–580°C day) indicating a faster rate of plant development at two experimental sites. The effect of ozone on chlorophyll content depended on the time and dose of O3 exposure. During grain filling, high O3 concentrations induced premature senescence of the flag leaves (up to −130°C day). This deleterious effect was mitigated by elevated CO2. Drought stress led to faster chlorophyll breakdown irrespective of CO2 treatment.