Neural differentiation of mouse embryonic stem cells in combination with three-dimensional electrospun nanofibers as an artificial extracellular matrix can be utilized to reconstruct a spinal cord defect. In this study, random and parallel-aligned nanofibrous poly ε-caprolactone was fabricated using electrospinning. Its hydrophobicity was modified by O2 plasma treatment to facilitate enhanced cell attachment. Embryoid bodies (EBs), which contain all three embryonic germ layers, were cultured on poly ε-caprolactone scaffolds to study the effect of fiber orientation on cell morphology and differentiation. Cell morphology and neuron-specific gene and protein expressions were, respectively, evaluated by scanning electron microscopy, real-time polymerase chain reaction, and immunocytochemistry. Although two types of nanofibrous scaffolds showed neural marker expression at the protein level, cells on randomly oriented scaffolds showed short-range topographical guidance and stretched across multiple directions, whereas cells on the parallel scaffolds exhibited long extension with enhanced neuron outgrowth along the fiber, producing oriented extracellular matrix, leading to direct cell migration and nerve regeneration. Quantitative real-time polymerase chain reaction showed that both aligned and random electrospun nanofibers downregulated the precursor neural marker Nestin compared with that in the control group, a gelatin-coated tissue culture plate (T). Analysis also showed higher expression of dorso-ventral neural markers (Isl1/2 and Lim1/2) than motor neuron progenitor markers (Pax6, Nkx6.1, and olig2) in aligned nanofibers than in the T group. Moreover, aligned nanofibers showed higher expression of mature neural specific markers such as β-tub and Map2 than those in the randomly oriented scaffolds. Therefore, we conclude that nanofibers with different orientations can support the neural lineage, but aligned nanofibrous scaffolds are superior candidates to promote the advancement of neural precursors to achieve maturity during the differentiation process.