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Different scale microstructures at different scales were obtained by one and two-step isothermal transformation. The deformation behavior and stability of retained austenite under the action of nanoindentation stress was investigated in these different microstructures. The results demonstrate that the boundary fraction and density of the K–S relationship increase significantly after two-step isothermal transformation, promoting the nucleation of bainite and resulting in a refined microstructure. Changes in the nano-hardness as a function of indentation depth and morphology confirmed the quantity of RA transformation and the composite deformation/transformation behavior of blocky martensite/retained austenite (M/RA). A higher dislocation density is enriched in RA with higher KAM with the two-step isothermal process. This resulted in higher stability to delay the occurrence of the pop-in phenomenon. The stability of RA is highly dependent on the crystal orientation, with (001) oriented RA having an increased propensity to undergo mechanical transformation to martensite, with additional pop-ins generated during loading.