||Engineering an artificial matrix to direct stem cell neuronal differentiation provides a promising approach for neural tissues repair and regeneration. Stem cells can serve as an ideal cell source due to their intrinsic ability to proliferate and produce specific cell lineage. They can be induced to differentiate into neurons under a permissive microenvironment. However, it remains unclear how to orchestrate various properties of the artificial matrix to achieve this objective most efficiently. In this project, we have considered multiple matrix properties and created an artificial extracellular matrix with designer self-assembling peptide (SAP) that favors neuronal differentiation. These properties include the presentation of biochemical signals, mechanical strength and culture dimensionality. We took advantage of the ease of using SAP to tailor different matrix properties to conduct a factorial analysis on stem cell proliferation and neuronal differentiation. The study has identified three-dimensional (3D) culture in a soft SAP matrix (with storage modulus from 262 to 672 Pa) presenting laminin-derived pentapeptide signal as the most favorable for neuronal differentiation for the P19 embryonic carcinoma. The results also revealed the synergistic effect between culture dimensionality and signal presentation. Optimal matrix properties identified with these pluripotent murine cells were also found applicable to totipotent murine embryonic stem cells. In absence of any growth factor, this 3D tailor-made matrix was found to promote the spontaneous formation of embryoid bodies (EBs) and prime the neuronal differentiation. During 3D culture, cells expressed a marker of neuronal differentiation but did not display characteristic neuronal morphology. When retrieved and re-cultured on a tissue culture plate (without SAP), the primed neuron like cells continued to differentiate, exhibiting extended neurite outgrowths and networks, elevated expression of genes of mature neurons (MAP2 and MeCP2), and down-regulation of glial marker (GFAP). In summary, an integrative and versatile approach for systematically studying cell-matrix interaction was developed and demonstrated in this study. By customizing the artificial matrix, our approach can be applied to investigate stem cells differentiation of other lineages, which will help further elucidate the complex cell-matrix interactions. It will provide more detailed guideline to generate the suitable artificial matrix for stem cell-specific differentiation.