Human natural killer cells originate from hematopoietic stem cells and can be differentiated in vitro through coculture with EL08.1D2 cells, a developmentally supportive stromal cell line. It is thought that these stroma support NK cell maturation by providing signaling cues and structural support that the stem cell would normally experience within tissue in vivo. These cues from the local microenvironment induce changes in cell motility and adhesion to guide cell fate. Although these signals are well studied in T and B lymphocytes, the full extent of these interactions is currently not well understood for NK cell development. In this dissertation, I describe changes in NK cell motility and adhesion that occur throughout development. Specifically, I show that developing NK cells undergo continuous changes in cell migration velocity and displacement that define their differentiation from precursor to functionally mature cell. In addition, I present extracellular matrix (ECM) derived from EL08.1D2 stroma as an alternate substrate for supporting NK cell development. This cell-derived matrix is composed of naturally secreted ECM components from the stroma and contains expected ECM components such as collagen and fibronectin. I show that this cell-derived matrix can support NK cell differentiation from hematopoietic precursor cells, and I further define the migratory and adhesive behaviors of NK developmental intermediates on EL08.1D2 and EL08.1D2-derived cell free matrix. Particularly, NK cell motility is acquired through development and NK cells undergo similar motility on both EL08.1D2 stroma and cell-derived matrix. The information presented in this work defines the extent to which developmentally supportive stroma are required for NK cell development in vitro and provides an avenue into research for alternative substrates and conditions for NK cell development.