Astronauts are exposed to radiation during space travel under conditions of dramatically reduced weightbearing activity. However, we know little about how gravity-dependent loading affects tissue sensitivity to radiation. We hypothesize gravity-dependent loading and irradiation share common molecular signaling pathways in bone cell progenitors that are sensitive to stress-induced reactive oxygen species (ROS), species capable of impacting skeletal health. To address this, progenitor cells with potential to differentiate into bone-forming osteoblasts were extracted from bone marrow, then cells were centrifuged (from 5-gravity (g) to 50-g for 5-180 min) on day 2 in culture, or were exposed to a single dose (1-5 Gy) of irradiation (137Cs 1 Gy/min) on day 3 or 4. Production of ROS was measured via fluorescence-activated cell sorting (FACS) using an oxidation-sensitive dye. Cell numbers were assessed by measurement of DNA content (CyQUANT). Osteoblastogenesis was estimated by measurement of alkaline phosphatase (ALP) activity and production of mineralized matrix (Alizarin Red staining). Transient centrifugation was a potent stimulus to bone marrow stromal cells, increasing production of ROS (1.2-fold), cell number (1.5-fold to 2.2-fold), and ALP activity (2.7-fold). Radiation also caused dose- and time-dependent increases in ROS production (1.1-fold to 1.4-fold) by bone marrow stromal cells, but inhibited subsequent osteoblast differentiation. In summary, gravity-dependent loading by centrifugation stimulated ROS production and increased numbers of osteoblasts. Although radiation increased production of ROS by bone marrow stromal cells, cell number and differentiation of osteoprogenitors appeared reduced. We conclude gravity-dependent loading and radiation both stimulate production of ROS and affect critical bone cell functions including growth and differentiation.