Osteoblast differentiation and migration are necessary for bone formation during bone remodeling. Mice lacking the proline-rich tyrosinekinase Pyk2 (Pyk2-KO) have increased bone mass, in part due to increased osteoblast proliferation. Megakaryocytes (MKs), the platelet-producing cells, also promote osteoblast proliferation in vitro and bone-formation in vivo via a pathway that involves Pyk2. In the currentstudy, we examined the mechanism of action of Pyk2, and the role of MKs, on osteoblast differentiation and migration. We found that Pyk2-KOosteoblasts express elevated alkaline phosphatase (ALP), type I collagen and osteocalcin mRNA levels as well as increased ALP activity, andmineralization, conﬁrming that Pyk2 negatively regulates osteoblast function. Since Pyk2 Y402 phosphorylation is important for its catalyticactivity and for its protein-scaffolding functions, we expressed the phosphorylation-mutant (Pyk2Y402F) and kinase-mutant (Pyk2K457A)inPyk2-KO osteoblasts. Both Pyk2Y402Fand Pyk2K457Areduced ALP activity, whereas only kinase-inactive Pyk2K457Ainhibited Pyk2-KOosteoblast migration. Consistent with a role for Pyk2 on ALP activity, co-culture of MKs with osteoblasts led to a decrease in the level ofphosphorylated Pyk2 (pY402) as well as a decrease in ALP activity. Although, Pyk2-KO osteoblasts exhibited increased migration compared towild-type osteoblasts, Pyk2 expression was not required necessary for the ability of MKs to stimulate osteoblast migration. Together, these datasuggest that osteoblast differentiation and migration are inversely regulated by MKs via distinct Pyk2-dependent and independent signalingpathways. Novel drugs that distinguish between the kinase-dependent or protein-scaffolding functions of Pyk2 may provide therapeuticspeciﬁcity for the control of bone-related diseases.