Constitutive activation of MEK1 and Akt together supports longer Epo-independent growth of erythroid progenitors than does H-ras.V12. Freshly isolated fetal liver TER119^- cells were infected with bicistronic retroviruses encoding GFP alone, H-ras.V12, ca.MEK and ca.Akt, ca.MEK and ca.Rlf, or ca.Rlf and ca.Akt, and cultured for 1 day. For double infection, one retroviral construct encodes hCD4 and the other encodes GFP. Doubly infected cells were stained for hCD4. Then, GFP⁺ cells (single infection) or GFP⁺hCD4⁺ cells (double infection) were sorted by FACS. Cells were then replated in fibronectin-coated wells in medium lacking Epo. Cell numbers are presented as total viable cells. Data are averages from triplicate cultures of a representative experiment and standard deviations were always less than 5% of the averages.