To understand further the effects of spaceflight on osteoblast-enriched cultures, normal chicken calvarial osteoblasts were flown aboard shuttle flight STS-77, and the total number of attached cells was determined. Spaceflight and control cultures were chemically fixed 3 h and 3 d after launch. These fixed cultures were processed for scanning electron microscopy (SEM). The SEM analysis showed that with just 3 d of exposure to spaceflight, coverslip cultures contained 300 ± 100 cells/mm², whereas 1G control samples contained a confluent monolayer of cells (2400 ± 200 cells/mm²). Although the cultures flown in space experienced a drastic decline in cell number in just 3 d, without further experimentation it was impossible to determine whether the decline was a result of microgravity, the harsh launch environment, or some combination of these factors. Therefore, this research attempted to address the effect of launch by subjecting osteoblasts to conditions simulating shuttle launch accelerations, noise, and vibrations. No differences, compared with controls, were seen in the number of total or viable cells after exposure to these various launch conditions. Taken together, these data indicate that the magnitude of gravitational loading (3G maximum) and vibration (7.83G rms maximum) resulting from launch does not adversely affect osteoblasts in terms of total or viable cell number immediately, but launch conditions, or the microgravity environment itself, may start a cascade of events that over several d contributes to cell loss.