The fatigue threshold behavior of stainless steel was assessed in high temperature air and hydrogenated deaerated water environments as a function of stress ratio (R). Fatigue threshold experiments were conducted on four different heats of type 304, 304/304L, and 308L austenitic stainless steels in 250°C air and water environments at stress ratios ranging from 0.1 to 0.8. Air and water experiments showed that operational threshold ΔK (ΔKTH) values ranged from 4.3–6.0 and 3.9–5.3 MPa√m, respectively. ΔKTH values were observed to generally decrease with increasing R which is attributable to crack closure effects. The water ΔKTH measurements were either consistent with or lower than air threshold measurements, and the potential roles of the competing effects of crack closure and hydrogen enhanced planar slip will be discussed in the context of these results. Load history effects in the form of overloads and underloads were shown to significantly impact ΔKTH measurements and these results motivated testing aimed at characterizing material property based intrinsic ΔK threshold (ΔKTH*) values. The ΔKTH* values for stainless steel fatigue crack growth in 250–288°C air and water environments are estimated to be 3 and 2 MPa√m, respectively.