The in vitro culture system outlined in this chapter allows for the delineation of events that occur during the development of CD34⁺ hematopoietic precursor cells into mature KIR⁺ human NK cells. This system can also be utilized to study the effects of gene overexpression or knockdown on the process of NK cell differentiation through retroviral transduction and long-term culture. The necessary soluble factors and contact-dependent conditions for in vitro human NK cell development have been worked out in our laboratory over the past 16 years.