Purpose: This study investigated the influence of smooth, roughened, and tricalcium phosphate (TCP)-coated roughened titanium-aluminum-vanadium (Ti-6Al-4V) surfaces on the osteogenic potential of rat bone marrow stromal cells (BMSCs). Methods: Machined smooth (MS), grit-blasted roughened (MT), and roughened surfaces coated with TCP were prepared from Ti-6Al-4V. Plastic surfaces were used as a control. Surface topography and chemical characteristics were determined. Cell attachment, morphology, proliferation, and temporal expression of mRNA and protein markers associated with bone healing were examined. Results: Roughness values were 0.09 ± 0.02 µm, 2.71 ± 0.24 µm, and 6.08 ± 0.62 µm for MS, MT, and TCP, respectively. Cell attachment was similar on all surfaces. The cell expansion phase occurred during days 1 to 3 on MS surfaces and days 3 to 5 on MT and TCP surfaces. The earlier onset of differentiation on MS surfaces versus MT and TCP surfaces was evidenced by: high mRNA expression peak for Runx2 at day 5 on MS (day 7 on MT and TCP); higher mRNA expression for osteopontin, osteonectin, bone sialoprotein (BSP), osteocalcin, type 1 collagen, and alkaline phosphatase over days 5 to 12 on MS compared with MT and TCP; higher levels of bone matrix proteins on MS compared with MT, with only BSP detected on TCP; cell morphology consistent with descriptions of differentiating osteoblasts apparent at day 5 on MS and absent on MT. Compared to plastic surfaces, Ti-6Al-4V appeared to suppress mRNA for interleukin 1ß, tumor necrosis factor alpha, and peroxisome proliferator-activated receptor gamma expression and upregulate osteoprotegerin. Conclusions: Cell expansion was delayed on roughened Ti-6Al-4V surfaces, impeding osteoblast differentiation and bone matrix synthesis. These results disagree with a number of published studies examining pure titanium. Ti-6Al-4V surfaces appear to assist in the resolution of proinflammatory cytokines and inhibit BMSC differentiation toward adipocytes. Keywords: calcium phosphate, osseointegration, osteoblasts, titanium