This study examined the effects of vacuum heat-treatment on the growth of oxide (thermally grown oxide, TGO) between the ceramic top coat (yttria-stabilized zirconia, YSZ) and the metallic bond coat (NiCrAlY) of a thermal barrier coating (TBC). For the investigation, IN738LC coupons coated with a TBC were heat-treated in vacuum and/or isothermally oxidized at 1200 °C and then microscopically analyzed using SEM and EDX. The introduction of heat treatment in vacuum before isothermal oxidation resulted in a reduction of the TGO thickness and the number of interface cracks around the TGO layers. These TGO layers were further analyzed using EDX line scan data in terms of the dependence of the EDX Al intensity on the O intensity. The analysis showed that the TGO layers in the coupons only oxidized in air were divided into two sub-layers with respect to the gradient of the Al intensity on the O intensity. The pre-treatment in vacuum nullified this division and reduced cracks at the interfaces. The effects of the treatment in vacuum on the behavior of TGO and TBCs were analyzed and compared with the data obtained for a TBC-coated turbine blade serviced for a land-based heavy duty gas turbine.