Body of Abstract: MAP kinase cascades are some of the most highly conserved cellular signaling complexes of eukaryotes. In the flowering plant Arabidopsis, a MAP kinase pathway including the MAP kinase YDA and the two MAP kinases MPK3 & MPK6 regulate cellular polarity and asymmetric division in embryonic development. The MAP kinase kinases MKK4 & MKK5 have also been implicated in this pathway, but analysis has been limited due to the lack of null alleles in the two genes. We have generated such alleles by genome editing. Analysis of the double mutant embryos in the progeny of plants mutant for the MKK5 allele and segregating for the MKK4 allele was accomplished by measuring embryo size at the two-cell stage as well as assessing further embryo development using DIC microscopy. Elongation of the zygote and the positioning of the first division plane are affected in a quantitatively similar fashion as in embryos lacking MPK3/MPK6. Double mutant embryos of both genotypes arrest as small, disorganized structures lacking a suspensor; in addition, less than the expected 25% mutants are produced. A PCR-based molecular marker was used to determine the transmission of double mutant gametes in reciprocal crosses. We found a normal Mendelian transmission rate within the female mutant gametophyte, but a reduced transmission rate within the male mutant gametophytes, suggesting the MKK4 and MKK5 genes together impact formation or growth of pollen in a similar fashion as MPK3/MPK6. The close overall similarity of mutant phenotypes strongly suggests that MKK4/MKK5 are the MAPK kinases acting upstream of the MAP kinases MPK3/MPK6 in pollen and early embryos.