Embryonic stem cells (ES cells) have been used extensively over the last decade to manipulate the murine genome. The vast majority of the modifications made have been in ES cells derived from the 129 mouse strain. Recently, various groups have successfully derived ES cells from a variety of inbred mouse strains. We report here the efficient derivation of germline competent ES cells from an outbred mouse model of human sickle cell disease. Knockout-transgenic sickle cell disease mice were produced by interbreeding mice with targeted deletions of their endogenous adult α- and β-globin genes with transgenic animals that synthesize human fetal hemoglobin during embryonic/fetal life and sickle hemoglobin after birth. This time-intensive breeding program resulted in mice that synthesize 100% human hemoglobin in their red blood cells (RBCs). After the first week of life, these sickle cell mice develop a severe hemolytic anemia with concomitant in vivo pathology similar to that seen in the human disease. Blastocysts were collected from hormonally-ovulated female sickle mice, which had been mated with male sickle mice and plated on primary embryonic fibroblast feeder cells (PEF). After six days in culture inner cell mass outgrowths were disaggregated and transferred onto fresh PEF. Fourteen individual lines of ES cells were derived from 139 blastocysts for an overall efficiency of 10%. Seventy-eight percent (11/14) of the cell lines maintained a full compliment of 40 chromosomes. Four out of the 11 cell lines were determined to be male by PCR with Y chromosome specific primers. Three male ES cell lines were injected into C57B1/6J blastocysts to examine their capacity for developmental competency. All 3 cell lines generated chimeric animals. High-level male chimeras from each line were mated to mice that synthesize 100% human HbA in their RBCs and each line exhibited germline transmission. These novel sickle ES cells will; (1) allow the study of the maturation of sickle erythroid progenitors and the testing of anti-sickling agents in culture after in vitro differentiation; (2) accelerate the generation of knockouts of disease modifying genes by eliminating the need for extensive mating and backcrossing; and (3) promote the development of cell therapy approaches for the correction of sickle cell disease.