Signaling molecules secreted during bone fracture healing stimulate molecular and cellular changes to promote healing and tissue regeneration. Molecular characterization of cytokines and growth factors can provide insight into their function and potential application as biologics or drug targets for bone tissue regeneration. Previous studies have identified the expression of IL1B, IL6, IL34, IGF1, ANGPT1, and TGFB3 during osteogenic differentiation and facture healing. In this study, we evaluated the transcriptome of adipose-derived mesenchymal stromal cells (AMSCs) following stimulation with these biological factors and examined their role in osteogenic differentiation. Stimulation of AMSCs with recombinant human (rh)-IL1B led to the largest gene expression changes compared to the other signaling factors including 1411 induced and 541 repressed genes (>2-fold). Rh-IL1B regulated genes included inflammatory (IL1B, IL6, CCL2) and osteogenic (BMP2, RUNX2, TWIST2) genes. Rh-IL1B also induced the expression of BMP2 in bone marrow-derived mesenchymal stromal cells (BMSCs) and bone digest cells. Although no changes in osteogenic differentiation of AMSCs was observed, low concentrations of rh-IL1B (0.1 ng/mL) promoted alkaline phosphatase activity of differentiated bone digest cells. Further, rh-IL1B significantly induced the secretion of BMP2 from these cells. Together this data provides insight into the molecular function of fracture-associated biological factors.