Assays that predict toxicity are an essential part of drug development. Many drugs fail in phase I clinical trials; therefore, there is a demand for models that can better predict human responses. A positive result in an efficient and predictive prescreen can prevent the wasted effort of further product development. The in vivo micronucleus (MN) assay is a robust toxicity test that assesses the genotoxic effect of drugs on adult bone marrow (BM) using the metric of genotoxic damage to the reticulocyte population in mice. An in vitro correlate to this assay might allow extension to human cells and thus better predictive power in drug development. A recently developed culture system that stimulates erythroid differentiation from Ter-119- fetal mouse liver offers the possibility to create an in vitro corollary to the in vivo MN assay. As first steps in developing a toxicity assay, we have (a) adapted this in vitro erythropoietic culture system to induce optimized erythropoietic growth from the Lineage-marker- (Lin-) population in adult BM, as adult hematopoietic tissue is ultimately a feasible source of cells; and (b) demonstrated that exposure to alklylating agents induces MN-formation in this culture system. The potential for increased efficiency in this in vitro model depends on the ability to stimulate terminal erythroid differentiation at an optimal level from adult BM. With these goals in mind, we have employed experimental design strategies and multi-linear regression to determine the sensitivity of Lin- BM to erythropoietic stimulation by relevant culture parameters, including Erythropoietin concentration, Stem Cell Factor concentration, oxygen tension, pH, and Fibronectin concentration, among others. From these studies, we were able to predict the erythropoietic response to a given growth condition (R squared = 0.9501) and we were able to quanitfy the relative size of key culture parameter effects on terminal erythropoietic growth. Further, through these stuides we have deonstrated that more than 1500 genotoxicity assays can be conducted using the BM of a single mouse. This throughput is significantly higher than that offered by the current in vivo assay, which assays a single condition per mouse. Finally, we have introduced three different chemotherapeutic alkylating agents [1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and methylmethane sulfonate (MMS)] into this culture system and have quantified significant global cytotoxic responses and concomitant increases in MN-formation within the resulting reticulocyte populations. This increase in MN frequency after exposure to genotoxic alkylating agents provides a clear signal of the clastogenic mechanism that likely induced hematopoietic toxicity. Thus, this culture system can serve to mimic the physiological activity of alkylating chemotherapeutics in the adult hematopoietic compartment.