It is known that adenosine triphosphate (ATP) is released from red blood cells (RBCs) due to various forms of stimulation such as deformation, pharmacological stimuli, and hypoxia. To date, these various stimuli have been investigated individually. Here, we have combined a microflow system capable of initiating deformation-induced release of ATP from the RBCs at various levels of hypoxia as measured by percent oxygen saturation in the RBC sample. When values of ATP released from deformation and hypoxia are compared to values of ATP release due to hypoxia alone, the relationship between the two stimuli can be deduced. Measurement of RBC-derived ATP with the well-known chemiluminescence assay employing luciferin/luciferase indicates that RBCs deoxygenated for 4 min released 1.84 ± 0.075 μM ATP. The largest decrease in oxygen saturation was found to be between 0 s (66.3% O₂ saturation) and 15 s (22.3% O₂ saturation). RBCs deoxygenated to a 22.3% O₂ saturation released 0.374 ± 0.011 μM ATP when pumped through the microflow system. This value is an increase from 0.281 ± 0.007 μM ATP in the presence of flow alone. The ATP release after exposure to hypoxia at 22.3% O₂ saturation was 0.381 ± 0.014 μM ATP, a value statistically equivalent to that of hypoxia and flow combined. These data suggest that, at an oxygen saturation point of around 25.0% or above, deformation contributes to ATP release from the RBC; however, beyond this saturation point, the ATP release is largely due to hypoxia.