A pushout test method was used to quantify effect of thermal cycling temperatures on the delamination toughness of an electron beam physical vapor deposited thermal barrier coating (EB-PVD TBC). The delamination toughness, Γi, was related to the maximum thermal cycling temperature, Th, equal to 1000, 1025, 1050, and 1100 °C. The measured delamination toughness varied from 9 to 95 J/m2. At Th = 1000 °C, Γi attained a maximum value, larger than that of the as-deposited sample and decreasing with increased Th. During the thermal cycling tests, the thermally grown oxide (TGO) was formed between the TBC and the bond coat deposited onto the superalloy substrate. Inside the TGO layer, mixture of Al2O3 and ZrO2 oxides was observed close to the TBC side with nearly pure Al2O3 phases close to the bond-coat side. During the pushout test, delamination occurred at the interface of the mixture and pure Al2O3 layer with an exception for Th = 1100 °C specimens where delamination also occurred at the interface between the TGO and bond-coat layers. The effect of thermal cycling temperatures on the delamination toughness is discussed in terms of the microstructural change and delamination behavior.