Hematopoietic stem cells (HSC) are inherently rare cell types that cannot be obtained in sufficient amounts for classical biochemical characterization. To facilitate functional studies of murine HSC and hematopoietic development, the technique of retroviral-mediated gene transfer provides a useful tool. The generation of high titer retroviral vectors permits transduction of stem cells with a variety of genes and leads to long-term marking in the blood of recipient mice. Optimized promoter/enhancers facilitate high-level transgene expression in mice transplanted with transduced bone marrow (BM) cells. The co-expression of reporter genes along with a gene of interest greatly facilitates tracking donor engraftment of transduced hematopoietic progeny following stem cell transplantation. This methodology can be used to reconstitute defective function in a mutant background or to study protein function during hematopoiesis by overexpression. Despite limitations such as integration site variegation and copy number-dependent effects, this approach is rapid and efficient compared with transgenic mouse technology. In this chapter, we review this broadly applicable technique for achieving high-level murine BM stem cell transduction. We also describe methods for transplantation and subsequent analysis of transplanted mice as a bona fide assay for the stem cell transduction efficiency.