Extracellular matrix (ECM) is a fibrous natural cell environment, possessing complicated micro- and nanoarchitectures, which provide extracellular signaling cues and influence cell behaviors. Mimicking this three-dimensional microenvironment in vitro is a challenge in developmental and disease biology. Here, suspended multilayer hierarchical nanofiber assemblies (diameter from micrometers to less than 100 nm) with accurately controlled fiber orientation and spacing are demonstrated as biological scaffolds fabricated using the non-electrospinning STEP (Spinneret based Tunable Engineered Parameter) fiber manufacturing technique. Micro/nanofiber arrays were manufactured with high parallelism (relative angles between fibers were maintained less than 6°) and well controlled interfiber spacing (<15%). Using these controls, we demonstrate a bottom up hierarchical assembly of suspended six layer structures of progressively reduced diameters and spacing from several polymer systems. We then demonstrate use of STEP scaffolds to study single and multicell arrangement at high magnifications. Specifically, using double layer divergent (0°-90°) suspended nanofibers assemblies, we show precise quantitative control of cell geometry (change in shape index from 0.15 to 0.57 at similar cell areas), and through design of scaffold porosity (80 × 80 μm(2) to 5 × 5 μm(2)) quadruple the cell attachment density. Furthermore, using unidirectional or crisscross patterns of sparse and dense fiber arrays, we are able to control the cell spread area from ∼400 to ∼700 μm(2), while the nucleus shape index increases from 0.75 to 0.99 with cells nearly doubling their focal adhesion cluster lengths (∼15 μm) on widely spaced nanofiber arrays. The platform developed in this study allows a wide parametric investigation of biophysical cues which influence cell behaviors with implications in tissue engineering, developmental biology, and disease biology.