The conventional biomimetic apatite coating process can be accelerated by immersing substrates into concentrated simulated body fluid (5× SBF) at 37°C to form an initial coating of apatite precursor spheres, and transform the precursors into plate-like apatite structures. Depending on processing parameters, different apatite structures can be created over the same substrate. The purpose of this study is to investigate the effects of the different apatite microenvironment on cell spreading, viability, proliferation, and gene expression. MC3T3-E1 preosteoblasts were cultured on five surfaces: conventional apatite (CA), precursor apatite spheres (PreA), large plate-like apatites (LgA), small plate-like apatites (SmA), and tissue culture grade polystyrene (TCPS). PreA induced significantly higher cell death during the first two weeks. TCPS supported more uniform spreading (1 day) and higher proliferation (2 weeks) than CA, LgA, and SmA. Apatites restricted spreading and promoted the extension of cellular projections along the textured surfaces under confocal microscopy observation. By 3 weeks, LgA induced highest expression of mature osteogenic markers osteocalcin (OCN) and bone sialoprotein (BSP) in both regular and osteogenic culture media based on quantitative real-time RT-PCR. The results of this study suggest differential cell responses to subtle changes in apatite microenvironment.