As well as other blood cells, B lymphocytes originate from hematopoietic stem cells. However, it is not fully understood how their production is controlled. In the serum-free, stromal-cell-free B cell differentiation culture described here, early steps of the B lineage differentiation process are reproduced under defined conditions. This assay is useful for examining the direct effects of various soluble factors on B cell progenitors because it does not contain stromal cells or unknown factors. Additionally, this assay yields sufficient cloning to measure B cell progenitors from single cell cultures. Stromal cell coculture assays are also described that cover a wider category of precursors such as human B cell progenitors.