Based on the hypothesis that precisely designed 3D microenvironments, in terms of 3D cell-cell/-matrix interactions and surface immobilized insoluble signaling factors, can control the HSC differentiation pathway, the overall goal of our study is to develop research tools for in-vitro HSC investigation. More specifically, the aim is to create 3D bone marrow and thymus niches for HSC differentiation into CD4 T cells. For this purpose, 3D geometry of inverted colloidal crystal (ICC) scaffold, which consists of empty spherical cavities arranged in a hexagonal crystal lattice and interconnecting channels between the cavities, was constructed with hydrogel matrix. Highly-interconnected pores facilitate cell transport, but their limited size and the number restrict free movement of floating cells simultaneously. Temporarily entrapped HSCs experience extensive cell-cell/-matrix interaction in a chamber. The complex 3D substrate was effectively modified utilizing a layer-by-layer (LBL) surface modification technique. The main driving force of a LBL method is the electrostatic interactions between two different components in aqueous solution, which is exceptionally useful for coating a 3D structure with biological molecules, with minimum loss of bioactivity. Five bi-layers of clay nanoparticles and poly(diallyldimethylammonium chloride) (PDDA) improved the cell adhesion on hydrophilic hydrogel matrix. Monolayer of Delta-like-1(DL-1) Notch ligand, one of essential insoluble signaling factors determining HSC developmental fate, was coated on top of Clay/PDDA multilayer. Preliminary results demonstrate that human HSCs were successfully cultured in the system. Surface marker characterization under a confocal microscope supports that LBL coated DL-1 ligand layer guided HSC differentiation pathway into CD4-T cell lineage. The results of this study will show that one can potentially simulate differentiation niches for different components of hematopoietic system.