We describe a method to derive cell lines and clones from cells of the murine midgestation aorta-gonads-mesonephros (AGM) microenvironment. We start from subdissected AGM regions in "explant" or "single cell suspension" type cultures from embryos transgenic for tsA58, a temperature-sensitive mutant of the SV40 T antigen gene. The number of cells in such cultures initially expand, but in most cases, this expansion phase is followed by a stable or even decline in cell number. After this so-called crisis phase, cell proliferation is noticeable in more than 90% of the cultures. Stromal cell clones can be isolated from these cultures, some of which have been cultured for more than 50 population doublings, and functionally characterized using various methods These stromal cell clones are valuable tools for the study of the regulation of hematopoietic stem and progenitor cells in the midgestation mouse embryo.